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The cell cycle is controlled by microtubule-associated serine/threonine kinase-like (MASTL), which phosphorylates the cAMP-regulated phosphoproteins 19 (ARPP19) at S62 and 19e/α-endosulfine (ENSA) at S67and converts them into protein phosphatase 2A (PP2A) inhibitors. Based on initial proteomic data, we hypothesized that the MASTL-ENSA/ARPP19-PP2A pathway, unknown until now in platelets, is regulated and functional in these anucleate cells. We detected ENSA, ARPP19 and various PP2A subunits (including seven different PP2A B-subunits) in proteomic studies of human platelets. ENSA-S109/ARPP19-S104 were efficiently phosphorylated in platelets treated with cAMP- (iloprost) and cGMP-elevating (NO donors/riociguat) agents. ENSA-S67/ARPP19-S62 phosphorylations increased following PP2A inhibition by okadaic acid (OA) in intact and lysed platelets indicating the presence of MASTL or a related protein kinase in human platelets. These data were validated with recombinant ENSA/ARPP19 and phospho-mutants using recombinant MASTL, protein kinase A and G. Both ARPP19 phosphorylation sites S62/S104 were dephosphorylated by platelet PP2A, but only S62-phosphorylated ARPP19 acted as PP2A inhibitor. Low-dose OA treatment of platelets caused PP2A inhibition, diminished thrombin-stimulated platelet aggregation and increased phosphorylation of distinct sites of VASP, Akt, p38 and ERK1/2 MAP kinases. In summary, our data establish the entire MASTL(like)-ENSA/ARPP19-PP2A pathway in human platelets and important interactions with the PKA, MAPK and PI3K/Akt systems., Competing Interests: The authors declare no conflict of interest.

Background: The glycoprotein (GP) Ib-IX-V complex is a unique platelet plasma membrane receptor, which is essential for platelet adhesion and thrombus formation. GPIbα, part of the GPIb-IX-V complex, has several physiological ligands such as von Willebrand factor (vWF), thrombospondin and distinct coagulation factors, which trigger platelet activation. Despite having an important role, intracellular GPIb-IX-V signaling and its regulation by other pathways are not well defined. Our aim was to establish the intracellular signaling response of selective GPIbα activation in human platelets, in particular the role of the tyrosine kinase Syk and its regulation by cAMP/PKA and cGMP/PKG pathways, respectively. We addressed this using echicetin beads (EB), which selectively bind to GPIbα and induce platelet aggregation., Methods: Purified echicetin from snake Echis carinatus venom was validated by mass spectrometry. Washed human platelets were incubated with EB, in the presence or absence of echicetin monomers (EM), Src family kinase (SFK) inhibitors, Syk inhibitors and the cAMP- and cGMP-elevating agents iloprost and riociguat, respectively. Platelet aggregation was analyzed by light transmission aggregometry, protein phosphorylation by immunoblotting. Intracellular messengers inositolmonophosphate (InsP1) and Ca 2+ i were measured by ELISA and Fluo-3 AM/FACS, respectively., Results: EB-induced platelet aggregation was dependent on integrin α IIb β 3 and secondary mediators ADP and TxA 2 , and was antagonized by EM. EB stimulated Syk tyrosine phosphorylation at Y352, which was SFK-dependent and Syk-independent, whereas Y525/526 phosphorylation was SFK-dependent and partially Syk-dependent. Furthermore, phosphorylation of both Syk Y352 and Y525/526 was completely integrin α IIb β 3 -independent but, in the case of Y525/526, was partially ADP/TxA 2 -dependent. Syk activation, observed as Y352/ Y525/Y526 phosphorylation, led to the phosphorylation of direct substrates (LAT Y191, PLCγ2 Y759) and additional targets (Akt S473). PKA/PKG pathways inhibited EB-induced platelet aggregation and Akt phosphorylation but, surprisingly, enhanced Syk and LAT/PLCγ2 tyrosine phosphorylation. A similar PKA/PKG effect was confirmed with convulxin-/GPVI-stimulated platelets. EB-induced InsP1 accumulation/InsP3 production and Ca 2+ -release were Syk-dependent, but only partially inhibited by PKA/PKG pathways., Conclusion: EB and EM are specific agonists and antagonists, respectively, of GPIbα-mediated Syk activation leading to platelet aggregation. The cAMP/PKA and cGMP/PKG pathways do not inhibit but enhance GPIbα-/GPVI-initiated, SFK-dependent Syk activation, but strongly inhibit further downstream responses including aggregation. These data establish an important intracellular regulatory network induced by GPIbα.

Platelets are circulating sentinels of vascular integrity and are activated, inhibited, or modulated by multiple hormones, vasoactive substances or drugs. Endothelium- or drug-derived NO strongly inhibits platelet activation via activation of the soluble guanylate cyclase (sGC) and cGMP elevation, often in synergy with cAMP-elevation by prostacyclin. However, the molecular mechanisms and diversity of cGMP effects in platelets are poorly understood and sometimes controversial. Recently, we established the quantitative human platelet proteome, the iloprost/prostacyclin/cAMP/protein kinase A (PKA)-regulated phosphoproteome, and the interactions of the ADP- and iloprost/prostacyclin-affected phosphoproteome. We also showed that the sGC stimulator riociguat is in vitro a highly specific inhibitor, via cGMP, of various functions of human platelets. Here, we review the regulatory role of the cGMP/protein kinase G (PKG) system in human platelet function, and our current approaches to establish and analyze the phosphoproteome after selective stimulation of the sGC/cGMP pathway by NO donors and riociguat. Present data indicate an extensive and diverse NO/riociguat/cGMP phosphoproteome, which has to be compared with the cAMP phosphoproteome. In particular, sGC/cGMP-regulated phosphorylation of many membrane proteins, G-proteins and their regulators, signaling molecules, protein kinases, and proteins involved in Ca 2+ regulation, suggests that the sGC/cGMP system targets multiple signaling networks rather than a limited number of PKG substrate proteins., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The cyclic nucleotides cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) regulate the activity of protein kinase A (PKA) and protein kinase G (PKG), respectively. This process helps maintain circulating platelets in a resting state. Here we studied the role of cAMP and cGMP in the regulation of megakaryocyte (MK) differentiation and platelet formation. Cultured, platelet-producing MKs were differentiated from fetal livers harvested from 13.5 days postcoital mouse embryos. MK development was accompanied by a dramatic increase in cAMP production and expression of soluble guanylate cyclase, PKG, and PKA as well as their downstream targets vasodilator-stimulated phosphoprotein (VASP) and MENA. Stimulation of prostaglandin E(1) receptor/adenylyl cyclase or soluble guanylate cyclase/PKG in cultured MKs increased VASP phosphorylation, indicating that these components share a common signaling pathway. To dissect out the role of cyclic nucleotides in MK differentiation, cAMP/PKA and cGMP/PKG signaling were alternately blocked in cultured MKs. Down-regulation of cAMP pathway effectors decreased MK numbers and ploidy. Notably, cGMP levels increased at the beginning of MK development and returned to basal levels in parallel with MK maturation. However, inhibition of cGMP pathway effectors had no effect on MK development. In addition, platelet release from mature MKs was enhanced by cGMP and inhibited by cAMP. Our data suggest that cAMP plays an important role in MK differentiation, while cAMP and cGMP have opposite effects on platelet production. Identifying the signaling pathways that underpin MK development and proplatelet formation will provide greater insights into thrombopoiesis and may potentially yield useful therapeutic targets., (Copyright © 2013 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

A number of direct thrombin inhibitors are successfully used clinically and experimentally as novel antithrombotics and specific anticoagulants. They are also used as anticoagulants in certain blood collection tubes for the analysis of platelet function. A series of platelet function tests have emerged to measure adequate responses to antiplatelet therapy. For comparative and practical reasons, it would be of advantage to use the same anticoagulant in blood collection tubes for different methods, e.g. thrombin inhibitors. However, there are little data on the effects of thrombin inhibitors on platelet signalling pathways that could influence results. We examined the applicability of thrombin inhibitor containing blood for platelet reactivity index (PRI) measurements of the VASP assay and investigated the effects of two thrombin inhibitors (hirudin and lepirudin) on cAMP- and cGMP-mediated signalling pathways in washed human platelets. We show that induction of VASP phosphorylation by PGE1 is markedly reduced in lepirudin containing blood samples. In consequence, PRI levels were highly variable compared to routinely used citrated blood. Surprisingly, in vitro incubation of platelets with thrombin inhibitors increases platelet cGMP levels and induces NOS independent sGC/PKG-mediated VASP phosphorylation. We conclude that thrombin inhibitors activate sGC/PKG-dependent pathways resulting in an increase of VASP phosphorylation which contributes to deviations in PRI measurements. These effects of thrombin inhibitors on sGC- and cGMP-mediated pathways including increased VASP phosphorylation may indicate the presence of an important additional platelet-based mechanism for the reduction of thrombus formation and thromboembolism by thrombin inhibitors.

Platelet activation is an irreversible process resulting in platelet apoptosis and necrosis, and circulating platelets contain many components of the apoptotic machinery. Cyclic guanosine monophosphate (cGMP) generated by nitric oxide (NO) activated soluble guanylyl cyclase (sGC) plays a crucial role in preventing platelet activation. However, in addition to activation of sGC, cGMP-independent NO effects in platelets have been described. To differentiate between cGMP-dependent and -independent NO effects on platelet apoptosis and reactive oxygen species (ROS) production, we generated platelet-specific sGC-deficient mice (PS-GCKO). Platelet apoptosis was induced by a combination of thrombin/convulxin (Thr/Cvx) and assessed by phosphatidylserine (PS) surface exposure, and loss of the mitochondrial membrane potential. NO-induced inhibition of PS externalisation was mediated only by cGMP-dependent mechanisms. Inhibition of the mitochondrial membrane potential decrease at low NO concentration was also cGMP-dependent but became cGMP-independent at high NO concentrations. In contrast, inhibition of ROS formation at any NO concentration was mediated by cGMP-independent mechanisms, very likely due to direct radical scavenging. NO inhibits platelet apoptosis by cGMP-dependent mechanisms and ROS production by cGMP-independent mechanisms. The PS-GCKO mouse model is an important tool for the differentiation of cGMP-dependent and -independent NO effects on platelets.

Platelets are specialized adhesive cells that play a key role in normal and pathological hemostasis through their ability to rapidly adhere to subendothelial matrix proteins (platelet adhesion) and to other activated platelets (platelet aggregation). NO plays a crucial role in preventing platelet adhesion and aggregation. In platelets, cGMP synthesis is catalyzed by sGC, whereas PDE2, PDE3 and PDE5 are responsible for cGMP degradation. Stimulation of cGK by cGMP leads to phosphorylation of multiple target substrates. These substrates inhibit elevation of intracellular calcium, integrin activation, cytoskeletal reorganization, and platelet granule secretion, events normally associated with platelet activation. The NO/cGMP pathway also plays a significant role in many other blood cell types in addition to platelets. In leukocytes, depending on the specific cell type, cGMP signaling regulates gene expression, differentiation, migration, cytokine production, and apoptosis.

Platelets play a crucial role in the physiology of primary hemostasis and pathophysiological processes such as arterial thrombosis. Accumulating evidence suggests a key regulatory role of both NO and reactive oxygen species (ROS) in platelets. While the inhibitory role of NO/cGMP signaling in both murine and human platelets is well established, recent data suggest that intracellular ROS generation is involved in platelet activation. Thrombin-induced intracellular ROS production was inhibited by NAD(P)H oxidase inhibitors (DPI and apocynin), cyclooxygenase inhibitor (acetylsalicylic acid), and superoxide scavengers (tiron and MnTMPyP). Furthermore, thrombin (Trap6)-induced platelet aggregation and thrombus formation on collagen under high shear was inhibited by NAD(P)H oxidase inhibitors (DPI and apocynin), whereas secretion and platelet shape change were not affected. Inhibition of alphaIIbbeta3 activation by NAD(P)H oxidase inhibitors and superoxide scavengers was independent of NO/cGMP signaling demonstrating a direct role of platelet NAD(P)H oxidase-generated ROS for integrin alphaIIbbeta3 activation.

Platelets play a crucial role in the physiology of primary hemostasis and pathophysiologic processes such as arterial thrombosis. Accumulating evidence suggests a role of reactive oxygen species (ROSs) in platelet activation. Here we show that platelets activated with different agonists produced intracellular ROSs, which were reduced by reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidase inhibitors and superoxide scavengers. In addition, we demonstrate that ROSs produced in platelets significantly affected alphaIIbbeta3 integrin activation but not alpha and dense granule secretion and platelet shape change. Thrombin-induced integrin alphaIIbbeta3 activation was significantly decreased after pretreatment of platelets with NAD(P)H oxidase inhibitors (diphenylene iodonium [DPI] [45% +/- 9%] and apocynin [43% +/- 11%]) and superoxide scavengers (tiron [60% +/- 9%] and Mn(III)tetrakis (1-methyl-4-pyridyl)porphyrin [MnTMPyP] [70% +/- 6%]). These inhibitors also reduced platelet aggregation and thrombus formation on collagen under high shear and achieved their effects independent of the nitric oxide/cyclic guanosine monophosphate (NO/cGMP) pathway.

Objective: Vasodilator-stimulated phosphoprotein (VASP) was identified as a substrate for cGMP-dependent protein kinase (PKG) and cAMP-dependent protein kinase (PKA). It is preferentially phosphorylated at serine239 by PKG, whereas serine157 is a preferred phosphorylation site for PKA. In addition, serine157 is phosphorylated by PKC in response to serum. We have investigated the effects of VASP and VASP phosphorylation at serine157 and serine239 on smooth muscle cell (SMC) proliferation and nitric oxide (NO)-mediated growth inhibition., Methods and Results: Aortic SMCs derived from VASP-deficient mice were transduced with retroviral vectors encoding either wild-type VASP or VASP mutants (S157A-VASP and S239A-VASP), in which serine157 and serine239, respectively, were replaced by a nonphosphorylatable amino acid, alanine. Expression of wt-VASP and S239A-VASP significantly increased proliferation, whereas expression of S157A-VASP was inhibitory. Expression of S239A-VASP rendered SMCs less sensitive to growth inhibition by the NO donor, S-nitroso-n-acetylpenicillamine, when compared with cells expressing wt-VASP. Similar effects were observed in cultured rat SMCs in which wt-VASP, S157A-VASP, and S239A-VASP were expressed., Conclusions: Our data suggest that VASP phosphorylation at serine157 is required for the growth-stimulatory effect of VASP in SMCs, whereas VASP phosphorylation at serine239 is involved in the growth inhibitory effects of NO on SMCs.

Platelets play a key role in hemostasis through their ability to rapidly adhere to activated or injured endothelium, subendothelial matrix proteins, and other activated platelets. A strong equilibrium between activating and inhibiting processes is essential for normal platelet and vascular function, impairment of this equilibrium being associated with either thrombophilic or bleeding disorders. Both cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) have been established as crucial and synergistic intracellular messengers that mediate the effects of platelet inhibitors such as nitric oxide (NO) and prostacyclin (PG-I2). However, it was recently suggested that a rapid cGMP/cGMP-dependent protein kinase (cGK)-mediated extracellular signal-related kinase (ERK) phosphorylation promotes platelet activation. This hypothesis was examined here by evaluating established and proposed cGK activators/inhibitors with respect to their capacity to promote either platelet activation or inhibition. In particular, the regulatory role of cGK for ERK phosphorylation and thrombin-, thromboxane-, and VWF-induced platelet activation was investigated. The data obtained do not support the concept that cGK-mediated ERK phosphorylation promotes platelet activation but confirm the inhibitory role of cGK in platelet function. One explanation for these discrepancies is the novel finding that extracellular cGMP analogs potently and rapidly inhibit thrombin-, thromboxane-, and VWF-induced human platelet signaling and activation by a cGK-independent mechanism.

Cardiac fibroblasts regulate tissue repair and remodeling in the heart. To quantify transcript levels in these cells we performed a comprehensive gene expression study using serial analysis of gene expression (SAGE). Among 110,169 sequenced tags we could identify 30,507 unique transcripts. A comparison of SAGE data from cardiac fibroblasts with data derived from total mouse heart revealed a number of fibroblast-specific genes. Cardiac fibroblasts expressed a specific collection of collagens, matrix proteins and metalloproteinases, growth factors, and components of signaling pathways. The NO/cGMP signaling pathway was represented by the mRNAs for alpha(1) and beta(1) subunits of guanylyl cyclase, cGMP-dependent protein kinase type I (cGK I), and, interestingly, the G-kinase-anchoring protein GKAP42. The expression of cGK I was verified by RT-PCR and Western blot. To establish a functional role for cGK I in cardiac fibroblasts we studied its effect on cell proliferation. Selective activation of cGK I with a cGMP analog inhibited the proliferation of serum-stimulated cardiac fibroblasts, which express cGK I, but not higher passage fibroblasts, which contain no detectable cGK I. Currently, our data suggest that cGK I mediates the inhibitory effects of the NO/cGMP pathway on cardiac fibroblast growth. Furthermore the SAGE library of transcripts expressed in cardiac fibroblasts provides a basis for future investigations into the pathological regulatory mechanisms underlying cardiac fibrosis.

Background and Purpose: Dipyridamole and in particular dipyridamole in combination with low-dose aspirin are very effective in preventing recurrent stroke. However, the mechanism(s) underlying this dipyridamole effect have not been elucidated. Since dipyridamole inhibits the cGMP-specific phosphodiesterase type V in vitro, we hypothesized and tested whether therapeutically relevant dipyridamole concentrations enhance NO/cGMP-mediated effects in intact human platelets studied ex vivo., Methods: Phosphorylation of vasodilator-stimulated phosphoprotein (VASP), an established marker of NO/cGMP effects in human platelets, was quantified by phosphorylation-specific antibodies and Western blots. Serotonin secretion and thromboxane synthase activity were determined by fluorometric quantification of derivatized serotonin and synthase products, respectively., Results: Endothelium-derived factors such as NO and prostaglandin I2 are known to elevate both cGMP and cAMP levels with concomitant platelet inhibition and VASP phosphorylation. In our in vitro experiments, therapeutically relevant concentrations (3.5 micromol/L) of dipyridamole amplified only cGMP-mediated VASP phosphorylation due to the NO donor sodium nitroprusside, but not cAMP-mediated effects. Furthermore, thromboxane synthase activity and serotonin secretion, events important for initial platelet activation, were inhibited by sodium nitroprusside, an effect also enhanced by dipyridamole, demonstrating the functional relevance of these observations. Finally, the ex vivo enhancement of NO/cGMP effects was also observed with platelets obtained from healthy volunteers treated with extended-release dipyridamole., Conclusions: Under therapeutically relevant conditions, dipyridamole enhances platelet inhibition by amplifying the signaling of the NO donor sodium nitroprusside. These data support the concept that enhancement of endothelium-dependent NO/cGMP-mediated signaling may be an important in vivo component of dipyridamole action.

The important role of cGMP and cGMP-dependent protein kinase (cGPK) for the inhibition of platelet activation and aggregation is well established and due to the inhibition of fundamental platelet responses such as agonist-stimulated calcium increase, exposure of adhesion receptors and actin polymerization. The diversity of cGMP binding proteins and their synergistic interaction with cAMP signaling in inhibiting platelets indicates that a variety of cGMP targets contribute to its antiplatelet action. Since stimulation of G(i)-proteins was recently shown to be essential for complete platelet activation/aggregation, the possibility that G(i)-signaling events are cGMP/cGPK targets was investigated. Thus, the effect of elevated cGMP levels and selective cGPK activation on purinergic and adrenergic receptor-evoked decrease of platelet cAMP content was closely examined. Experiments with a selective activator of cGPK demonstrate for the first time a cGMP-caused G(i)-protein inhibition and our data suggest that this effect is mediated by cGPK. Considering the essential role of G(i)-signaling for platelet activation, we propose that inhibition of G(i)-mediated signaling by cGMP/cGPK is an important mechanism of action underlying the platelet inhibition by cGMP-elevating endothelium derived factors and drugs.

Background: In experimental animal models, long-term in vivo treatment with nitroglycerin (NTG) induces both endothelial dysfunction and tolerance to nitrates. However, it is still controversial whether nitrate tolerance in humans is associated with both endothelial dysfunction and impaired vascular response to nitrovasodilator-derived NO., Methods and Results: Patients undergoing elective bypass surgery were randomized to receive 48 hours of continuous NTG infusion (NTG group) or no nitrate therapy (control group). Segments of surgically removed arteria mammaria, vena saphena, and arteria radialis not required for the bypass procedure were used to examine (1) the vascular responsiveness to NTG and the endothelium-dependent vasodilator acetylcholine; (2) the expression of the NO target, the soluble guanylyl cyclase; (3) the expression of the soluble guanylyl cyclase/cGMP effector target, the cGMP-dependent protein kinase (cGK); and (4) the cGK activity as assessed by the phosphorylation state of its vascular substrate, the vasodilator-stimulated phosphoprotein at serine(239) (P-VASP). NTG treatment caused a marked degree of nitrate tolerance in all 3 vessel types studied and a significant cross-tolerance to the endothelium-dependent vasodilator acetylcholine in A. mammaria and A. radialis. Although soluble guanylyl cyclase, cGK-I, and VASP expression levels were not modified by NTG treatment, a marked decrease of P-VASP, a surrogate parameter for in-vivo cGK-I activity, was observed., Conclusions: We conclude that long-term NTG treatment induces endothelial dysfunction and impaired vascular NO/cGMP signaling in humans, which can be monitored by measuring P-VASP levels.

Angiotensin II infusion causes endothelial dysfunction by increasing NAD(P)H oxidase-mediated vascular superoxide production. However, it remains to be elucidated how in vivo angiotensin II treatment may alter the expression of the gp91(phox) isoforms and the endothelial nitric oxide synthase (NOS III) and subsequent signaling events and whether, in addition to the NAD(P)H oxidase, NOS III contributes to vascular superoxide formation. We therefore studied the influence of in vivo angiotensin II treatment (7 days) in rats on endothelial function and on the expression of the NAD(P)H oxidase subunits p22(phox), nox1, nox4, and gp91(phox) and NOS III. Further analysis included the expression of NO-downstream targets, the soluble guanylyl cyclase (sGC), the cGMP-dependent protein kinase I (cGK-I), and the expression and phosphorylation of the vasodilator-stimulated phosphoprotein (VASP) at Ser239 (P-VASP). Angiotensin II caused endothelial dysfunction and increased vascular superoxide. Likewise, we found an increase in vascular protein kinase C (PKC) activity, in the expression of nox1 (6- to 7-fold), gp91(phox) (3-fold), p22(phox) (3-fold), NOS III mRNA, and protein. NOS-inhibition with N(G)-nitro-L-arginine decreased superoxide in vessels from angiotensin II-treated animals, compatible with NOS-uncoupling. Vascular NO assessed with electron paramagnetic resonance was markedly reduced. Likewise, a decrease in sGC-expression and P-VASP levels was found. In vivo PKC-inhibition with chelerythrine reduced angiotensin II-induced superoxide production and markedly inhibited upregulation of NAD(P)H oxidase subunits. We therefore conclude that angiotensin II-induced increases in the activity and the expression of NAD(P)H oxidase are at least in part PKC-dependent. NADPH oxidase-induced superoxide production may trigger NOS III uncoupling, leading to impaired NO/cGMP signaling and to endothelial dysfunction in this animal model. The full text of this article is available at http://www.circresaha.org.

Cardiovascular diseases are often accompanied and aggravated by pathologic platelet activation. Tight regulation of platelet function is an essential prerequisite for intact vessel physiology or effective cardiovascular therapy. Physiological platelet antagonists as well as various pharmacological vasodilators inhibit platelet function by activating adenylyl and guanylyl cyclases and increasing intracellular cyclic AMP (cAMP) and cyclic GMP (cGMP) levels, respectively. Elevation of platelet cyclic nucleotides interferes with basically all known platelet activatory signaling pathways, and effectively blocks complex intracellular signaling networks, cytoskeletal rearrangements, fibrinogen receptor activation, degranulation, and expression of pro-inflammatory signaling molecules. The major target molecules of cyclic nucleotides in platelets are cyclic nucleotide-dependent protein kinases that mediate their effects through phosphorylation of specific substrates. They directly affect receptor/G-protein activation and interfere with a variety of signal transduction pathways, including the phospholipase C, protein kinase C, and mitogen-activated protein kinase pathways. Regulation of these pathways blocks several steps of cytosolic Ca(2+) elevation and controls a multitude of cytoskeleton-associated proteins that are directly involved in organization of the platelet cytoskeleton. Due to their multiple sites of action and strong inhibitory potencies, cyclic nucleotides and their regulatory pathways are of particular interest for developing new approaches for the treatment of thrombotic and cardiovascular disorders.

Studies with cGMP-dependent protein kinase I (cGK-I)-deficient human cells and mice demonstrated that cGK-I ablation completely disrupts the NO/cGMP pathway in vascular tissue, which indicates a key role of this protein kinase as a mediator of the NO/cGMP action. Analysis of the vasodilator-stimulated phosphoprotein phosphorylated at serine 239 (P-VASP) is a useful tool to monitor cGK-I activation in platelets and cultured endothelial and smooth muscle cells. Therefore, we investigated whether endothelial dysfunction and/or vascular NO bioavailability is reflected by decreased vessel wall P-VASP and whether improvement of endothelial dysfunction restores this P-VASP. Incubation of aortic tissue from New Zealand White Rabbits with the NOS inhibitor N:(G)-nitro-Ld-arginine and endothelial removal strikingly reduced P-VASP. Oxidative stress induced by inhibition of CuZn superoxide dismutase increased superoxide and decreased P-VASP. Endothelial dysfunction in hyperlipidemic Watanabe rabbits (WHHL) was associated with increased vascular superoxide and with decreased P-VASP. Treatment of WHHL with AT(1) receptor blockade improved endothelial dysfunction, reduced vascular superoxide, increased vascular NO bioavailability, and increased P-VASP. Therefore, the level of vessel P-VASP closely follows changes in endothelial function and vascular oxidative stress. P-VASP is suggested to represent a novel biochemical marker for monitoring the NO-stimulated sGC/cGK-I pathway and endothelial integrity in vascular tissue.

There is contradictory information on the relevance of nitric oxide (NO) and cGMP for the function of brain capillary endothelial cells (BCEC) forming the blood-brain barrier (BBB). Therefore, NO/cGMP-mediated signal transduction was investigated in cell cultures of BCEC and of astrocytes (AC) inducing BBB properties in BCEC. Constitutive, Ca2+-activated isoforms of NO synthase (NOS) were found in BCEC (endothelial NOS: eNOS) and in AC (neuronal NOS: nNOS), leading to increased NO release after incubation with the Ca2+-ionophore A23187. Both cell types expressed inducible NOS (iNOS) after incubation with cytokines. Soluble guanylate cyclase (sGC) was detected in both cell types. NO-dependent cGMP formation were observed in BCEC and, less pronounced, in AC. Furthermore, both cell types formed cGMP independently of NO via stimulation of particulate guanylate cyclase (pGC). cGMP-dependent protein kinase (PKG) type Ibeta, but not type II, was expressed in BCEC and AC. In BCEC, vasodilator-stimulated phosphoprotein (VASP) was detected, an established substrate of PKG and associated with microfilaments and cell-cell contacts. Phosphorylation of VASP was intensified by increased intracellular cGMP concentrations. The results indicate that BCEC and, to a smaller degree, AC can form NO and cGMP in response to different stimuli. In BCEC, NO/cGMP-dependent phosphorylation of VASP is demonstrated, thus providing a possibility of influencing cell-cell contacts., (Copyright 1999 Elsevier Science B.V.)

Nitric oxide (NO), the physiological activator of soluble guanylyl cyclase (sGC), induces inhibitory effects on platelet activation via elevation of cGMP levels and stimulation of the cGMP-dependent protein kinase. YC-1, a benzylindazole derivative, was shown to activate sGC in intact platelets, resulting in inhibition of platelet aggregation. In a previous study, we demonstrated that YC-1 not only stimulates purified sGC but also potentiates the stimulatory action of submaximally effective NO and carbon monoxide (CO) concentrations. Here, we investigated the potentiating effect of YC-1 in intact platelets. YC-1 together with NO or CO led to complete inhibition of platelet aggregation at concentrations that were ineffective by themselves. Maximally effective 2, 2-diethyl-1-nitroso-oxyhydrazine (3 microM) and YC-1 (100 microM) concentrations each elevated the cGMP levels in intact platelets approximately 13-fold, and administration of the two drugs together resulted in enormous potentiation of cGMP formation, which greatly exceeded the effect on the purified enzyme and yielded a >1300-fold increase in cGMP levels. Similar results were obtained using CO instead of NO. Furthermore, YC-1 not only stimulated sGC but also inhibited cGMP-hydrolyzing phosphodiesterases in platelets. The enormous elevation of cGMP levels led to enhanced phosphorylation of the cGMP-dependent protein kinase substrate vasodilator-stimulated phosphoprotein. Thus, by the combination of two effects (i.e., potentiation of NO-induced sGC stimulation and phosphodiesterase inhibition), YC-1-like substances are potent activators of the sGC/cGMP pathways and are therefore interesting candidates to act as modulators of cGMP-mediated effects, especially within the cardiovascular system.

NO and cGMP have emerged as important signal transduction mediators of the effects of certain hormones, inter-/intracellular signals, toxins and drugs. However, a major challenge is to define relevant criteria for determining which of the many NO and/or cGMP effects are dependent on cGMP-dependent protein kinases (cGKs). Important criteria include that: (1) the cell types/tissues investigated contain at least one form of cGK which is activated by the cGMP-elevating agent in the intact cell system; (2) specific activators/inhibitors of cGKs mimic/block the effects of cGMP-elevating agents in the intact cell system; and (3) the cGMP effect is absent or blunted in cGK-deficient systems, or can be reconstituted by the introduction of active cGKs. Previously, analysis of cGK activity in intact cells has been very difficult. However, the analysis of vasodilator-stimulated phosphoprotein (VASP) phosphorylation by polyclonal antibodies and newly developed monoclonal antibodies, each of which specifically recognize different phosphorylation sites, allows the quantitative measurement of cGK activity in intact cells. With the use of these methods, the properties of certain cGK mutants, cGK activators (cGMP, 8-Br-cGMP, 8-pCPT-cGMP) as well as various "specific cGK inhibitors" (KT 5823, Rp-8Br-PET-cGMPS, Rp-8-pCPT-cGMPS, H8 and H89) were investigated. Although these "specific cGK inhibitors" have been widely used to establish or rule out functional roles of cGKs, very few studies have actually addressed the efficiency/specificity of such compounds in intact cells. Our results demonstrate that these inhibitors are useful tools only when used in combination with other experimental approaches and biochemical evidence.

In order to investigate the involvement of cGMP-dependent protein kinase (cGK) type II in cGMP-provoked intestinal Cl- secretion, cGMP-dependent activation and phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels was analyzed after expression of cGK II or cGK Ibeta in intact cells. An intestinal cell line which stably expresses CFTR (IEC-CF7) but contains no detectable endogenous cGK II was infected with a recombinant adenoviral vector containing the cGK II coding region (Ad-cGK II) resulting in co-expression of active cGK II. In these cells, CFTR was activated by membrane-permeant analogs of cGMP or by the cGMP-elevating hormone atrial natriuretic peptide as measured by 125I- efflux assays and whole-cell patch clamp analysis. In contrast, infection with recombinant adenoviruses expressing cGK Ibeta or luciferase did not convey cGMP sensitivity to CFTR in IEC-CF7 cells. Concordant with the activation of CFTR by only cGK II, infection with Ad-cGK II but not Ad-cGK Ibeta enabled cGMP analogs to increase CFTR phosphorylation in intact cells. These and other data provide evidence that endogenous cGK II is a key mediator of cGMP-provoked activation of CFTR in cells where both proteins are co-localized, e. g. intestinal epithelial cells. Furthermore, they demonstrate that neither the soluble cGK Ibeta nor cAMP-dependent protein kinase are able to substitute for cGK II in this cGMP-regulated function.

The mechanism underlying the synergistic inhibition of platelet activation by cGMP- and cAMP-elevating vasodilators was investigated using washed human platelets and platelet-rich plasma. With both types of human platelet preparations, low concentrations of sodium nitroprusside increased the cAMP-elevating potency of low concentrations of prostaglandin E1 (PG-E1). Using threshold concentrations of both sodium nitroprusside and PG-E1, the NO-donor potentiated the effect of PG-E1 with respect to the phosphorylation of the focal adhesion-associated vasodilator-stimulated phosphoprotein (VASP) at serine157. In contrast, threshold concentrations of cell-membrane permeant selective activators of the platelet cGMP-dependent protein kinase or the cAMP-dependent protein kinase had only additive effects on VASP serine157 phosphorylation in washed human platelets. The data demonstrate that low intracellular levels of cGMP effectively inhibit type III cGMP-inhibited phosphodiesterase in human platelets despite the high levels of cGMP-dependent protein kinase present in this cell type. This study provides the first evidence that the simultaneous activation of both cGMP- and cAMP-dependent protein kinase results in additive effects on VASP serine157 phosphorylation, whereas the supra-additive effects observed with the combination of sodium nitroprusside and PG-E1 are due to cGMP-mediated inhibition of type III phosphodiesterase. VASP phosphorylation at serine157 may be an important component underlying the synergistic inhibition of human platelets by cGMP-and cAMP-elevating agents.

The presence and functional role of the cyclic nucleotide signal transduction system was investigated in platelets from patients with myeloproliferative disorders. Platelets from certain patients with chronic myelocytic leukemia showed decreased expression of cGMP-dependent protein kinase, and platelets from two such patients were studied in some detail. These platelets had very little if any cGMP-dependent protein kinase but a normal level of cAMP-dependent protein kinase. They also contained a normal level of VASP (vasodilator-stimulated phosphoprotein, a specific substrate of both cAMP- and cGMP-dependent protein kinase), as well as a functionally intact prostaglandin E1-stimulated cAMP-mediated VASP phosphorylation. In contrast, sodium nitroprusside-stimulated VASP phosphorylation was severely impaired in these cGMP-dependent protein kinase-deficient platelets, despite an exaggerated cGMP response to sodium nitroprusside. Furthermore, whereas selective activation of the cGMP-dependent protein kinase by 8-(4-chlorophenylthio)-cGMP strongly inhibited the ADP- or thrombin-evoked calcium mobilization from intracellular stores in normal platelets, this agonist-evoked calcium response was not inhibited by the cGMP analog in cGMP-dependent protein kinase-deficient platelets. The results demonstrate a defect in the nitrovasodilator-/cGMP-regulated signal transduction system in human platelets from some patients with myeloproliferative disorders, and underscore that a cGMP-dependent protein kinase regulatory system, distinct from that of cAMP-dependent protein kinase or other cGMP-dependent effectors is operative in normal human platelets.

The activation of human platelets is inhibited by two intracellular pathways regulated by either cGMP- or cAMP-elevating agents. There is considerable evidence that the inhibitory effects of cGMP and cAMP are mediated by the cGMP-PK and cAMP-PK, respectively, in human platelets. The cGI-PDE is an additional target for cGMP, and the cGMP-mediated elevation of cAMP levels contributes to the well known synergism between cAMP- and cGMP-elevating platelet inhibitors. Stimulation of both cAMP-PK and cGMP-PK prevents the agonist-induced activation of MLCK and PKC and inhibits the agonist-induced calcium mobilization from intracellular stores without any major effect on the ADP-regulated cation channel. These studies suggest that the inhibition of an early event of platelet activation, e.g. activation of PLC, is an effect common to both cGMP-PK and cAMP-PK stimulation. A common substrate of both cGMP-PK and cAMP-PK, the 46/50 kDa protein VASP, has been recently identified as a novel microfilament- and focal contact-associated protein whose phosphorylation correlates very well with platelet inhibition. Future investigations will have to identify the precise molecular mechanism of cyclic nucleotide inhibition of Ca2+ discharge from intracellular stores and whether cGMP-PK- and cAMP-PK-mediated VASP phosphorylation is an important component of this effect of cyclic nucleotides in human platelets.

8-(p-Chlorophenylthio)-cGMP (8-pCPT-cGMP) and 8-bromo-cGMP were compared with respect to their chemical and biological properties in order to evaluate their potential as selective activators of cGMP-dependent protein kinase (cGMP-PK; EC 2.7.1.37) in intact human platelets. 8-pCPT-cGMP, 8-Br-cGMP and cGMP were shown to be potent and selective activators of purified bovine lung cGMP-PK and of cGMP-PK present in human platelet membranes when compared with the activation of cAMP-dependent protein kinase (cAMP-PK; EC 2.7.1.37). 8-pCPT-cGMP was not hydrolysed by the purified cGMP-stimulated phosphodiesterase (cGS-PDE), cGMP-inhibited phosphodiesterase (cGI-PDE) and Ca(2+)-calmodulin-dependent phosphodiesterase (CaM-PDE), whereas cGMP and, to a lesser extent, 8-Br-cGMP were hydrolysed by all three types of 3',5' cyclic nucleotide phosphodiesterases (EC 3.1.4.17) examined. Also, 8-pCPT-cGMP was not hydrolysed by a human platelet homogenate which contains a high level of the cGMP-specific cGMP-binding phosphodiesterase (cGB-PDE). Additionally, 8-pCPT-cGMP did not activate the cGS-PDE or inhibit the cGI-PDE, whereas half-maximal inhibition of cGI-PDE occurred at 8 microM 8-Br-cGMP. The apparent lipophilicity of 8-pCPT-cGMP was higher than that of 8-Br-cGMP. Extracellular application of 8-pCPT-cGMP to intact human platelets reproduced the pattern of protein phosphorylation induced by sodium nitroprusside (SNP), a cGMP-elevating inhibitor of platelet activation. Quantitatively, 8-pCPT-cGMP was more effective than 8-Br-cGMP in inducing phosphorylation of the 46/50 kDa vasodilator-stimulated phosphoprotein, a major substrate of cGMP-PK in intact platelets. As observed with SNP, pretreatment of human platelets with 8-pCPT-cGMP prevented the aggregation induced by thrombin. The results suggest that 8-pCPT-cGMP is a very potent and selective activator of cGMP-PK in cell extracts and in intact human platelets and, in this respect, is superior to 8-Br-cGMP and other cGMP analogs used for intact cell studies. The data also suggest that inhibition of platelet activation in intact human platelets by nitrovasodilators is mediated by cGMP-PK.

Vasodilators capable of elevating cAMP or cGMP inhibit the activation of human platelets and stimulate the phosphorylation of a 46-kDa protein (vasodilator-stimulated phosphoprotein, VASP) mediated by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG). The availability of purified proteins and specific antisera against VASP, PKG and the catalytic subunit of PKA enabled us to measure and estimate the concentration of these regulatory proteins in intact human platelets. In addition, the rate of PKA- and PKG-mediated VASP phosphorylation in intact human platelets was estimated. For these calculations, a homogeneous population of human platelets and a homogeneous intracellular distribution of proteins and second messengers was assumed. Unstimulated washed human platelets contain 4.4 microM cAMP and 3.1 microM catalytic subunit of PKA, which is equivalent to 6.2 microM cAMP-binding sites due to PKA. Unstimulated washed human platelets also contain 0.4 microM cGMP and 7.3 microM PKG monomer, equivalent to 14.6 microM cGMP-binding sites due to the PKG. The intracellular concentration of VASP in platelets was estimated to be 25 microM. Treatment of washed human platelets with 10 microM (or 10 mM) prostaglandin E1 (PGE1) elevated the intracellular cAMP concentration to 27 microM (10 microM with 10 nM PGE1) within 30 s, accompanied by a rapid, up to 55% (35%), conversion of VASP from the dephosphorylated form (46-kDa protein) to the phosphorylated form (50-kDa protein). Treatment of washed human platelets with 100 microM (or 1 microM) sodium nitroprusside elevated the platelet cGMP level to 4 microM (0.9 microM with 1 microM sodium nitroprusside) within 2 min, accompanied by a less-rapid VASP phosphorylation of 45% (27% with 1 microM sodium nitroprusside). PGE1 and sodium nitroprusside had no significant effect on human platelet cGMP or cAMP levels, respectively. The results suggest for human platelets that relatively small increase in cAMP levels are required for activation of most of PKA, whereas even several-fold increases in platelet cGMP levels are capable of stimulating only a small fraction of total PKG. This interpretation was also supported by phosphorylation experiments with purified VASP, PKG and catalytic subunit of PKA. The results also support the hypothesis that in human platelets both cAMP/PKA- and cGMP/PKG-regulated VASP phosphorylation are components of an efficient and sensitive signal-transduction pathway, most likely involved in the inhibition of platelet activation.(ABSTRACT TRUNCATED AT 400 WORDS)

Most platelet agonists activate and elevate the cytosolic free calcium concentration in human platelets through receptor-dependent mechanisms that are antagonized by cAMP- and cGMP-elevating agents. Nitrovasodilators such as nitroprusside and endothelium-derived relaxing factor are potent cGMP-elevating platelet inhibitors. In the present study, the role of cGMP and cGMP-dependent protein kinase in nitrovasodilator inhibition of ADP- and thrombin-evoked calcium elevation and activation of human platelets was investigated. Preincubation of platelets with 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (8-pCPT-cGMP; a membrane-permeant selective activator of the cGMP-dependent protein kinase that does not significantly affect cGMP-regulated phosphodiesterases) inhibited the thrombin-induced phosphorylation mediated by myosin light chain kinase and protein kinase C. Nitrovasodilator-induced protein phosphorylation in human platelets was distinct from that induced by cAMP-elevating prostaglandins and could be mimicked by 8-pCPT-cGMP. Preincubation of human platelets with nitrovasodilators or 8-pCPT-cGMP inhibited the ADP- and thrombin-evoked calcium elevation in the presence and absence of external calcium. Nitrovasodilators and 8-pCPT-cGMP also inhibited the agonist-induced Mn2+ influx, but stopped-flow experiments indicated that the ADP receptor-operated cation channel was not significantly inhibited. These results suggest that in human platelets nitrovasodilators inhibit the agonist-induced calcium mobilization from intracellular stores and the secondary store-related calcium influx but not the ADP receptor-operated cation channel. The results also suggest that these nitrovasodilator effects are mediated by cGMP and the cGMP-dependent protein kinase.

We reported previously that a 46/50-kDa membrane-associated vasodilator-stimulated phosphoprotein (VASP) is phosphorylated in intact human platelets in response to both cGMP- and cAMP-elevating vasodilator drugs and presented evidence that this is mediated by cGMP- and cAMP-dependent protein kinases, respectively. VASP was recently purified and an antibody against it was developed which detects a phosphorylation-induced mobility change of VASP in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Halbrügge, M., Friedrich, C., Eigenthaler, M., Schanzenbächer, P., and Walter, U. (1990) J. Biol. Chem. 265, 3088-3093). We have now used these methods for the quantitative analysis of VASP phosphorylation during coincubations of human endothelial cells and human platelets. Endothelial cell-derived factors caused the rapid, stoichiometric, and reversible phosphorylation of platelet VASP during these coincubations. Other experiments indicated that the endothelium-derived factors which stimulate VASP phosphorylation are prostacyclin and endothelium-derived relaxing factor whose effects are mediated by cAMP/cAMP-dependent protein kinase and cGMP/cGMP-dependent protein kinase, respectively. The results suggest that VASP phosphorylation is an important component of the inhibitory effects of prostacyclin and endothelium-derived relaxing factor on platelet activation and that VASP phosphorylation is a useful biochemical marker for the interaction of endothelial cells and platelets.

Regulation of cardiac contraction by neurotransmitters and hormones is often correlated with regulation of the L-type Ca2(+)-channel current (ICa) through the opposite actions of two second messengers, cyclic AMP and cyclic GMP. While cyclic AMP stimulation of ICa is mediated by the activation of cyclic AMP-dependent protein kinase, inhibition of ICa by cyclic GMP in frog heart is largely mediated by activation of cyclic AMP phosphodiesterase. The present patch-clamp study reveals that, in rat ventricular cells, cyclic GMP can also regulate ICa via activation of endogenous cyclic GMP-dependent protein kinase (cGMP-PK). Indeed, the effect of cyclic GMP on ICa was mimicked by intracellular perfusion with the proteolytic active fragment of purified cGMP-PK. Moreover, cGMP-PK immunoreactivity was detected in pure rat ventricular myocytes by using a specific polyclonal antibody. These results demonstrate a dual mechanism for the inhibitory action of cyclic GMP in heart, as well as a physiological role for cGMP-PK in the control of mammalian heart function.

The activation of the cGMP-dependent protein kinase and cAMP-dependent protein kinase by the diastereomers of guanosine 3',5'-monophosphorothioate, (Sp)-cGMPS and (Rp)-cGMPS, and 8-chloroguanosine 3',5'-monophosphorothioate, (Sp)-8-Cl-cGMPS and (Rp)-8-Cl-cGMPS, was investigated using the peptide Kemptide as substrate. The (Sp)-diastereomers, which have an axial exocyclic sulfur atom, bound to the cGMP-dependent protein kinase and stimulated its phosphotransferase activity. In contrast, the (Rp)-isomers, which have an equatorial exocyclic sulfur atom, bound to the enzyme without stimulation of its activity. (Rp)-cGMPS and (Rp)-8-Cl-cGMPS antagonized the activation of the cGMP-dependent protein kinase with a Ki of 20 microM and 1.5 microM, respectively. (Rp)-cGMPS also antagonized the activation of cAMP-dependent protein kinase with a Ki of 20 microM. In contrast, (Rp)-8-cGMPS ws a weak inhibitor of the cAMP-dependent protein kinase with a Ki of 100 microM. (Rp)-8-Cl-cGMPS appears to be a rather selective inhibitor of the cGMP-dependent protein kinase and may be a useful tool for studying the role of cGMP in broken and intact cell systems.

A guanosine 3':5'-monophosphate (cGMP)-dependent protein kinase was purified from bovine lung using 8-(6-aminohexylamino)-cAMP-Sepharose. The activity of the purified enzyme was highly dependent on cGMP using histone f2b as a substrate. The self-phosphorylation of the purified enzyme was strongly inhibited by cGMP and not significantly affected by cAMP. A precipitating antiserum prepared in rabbits against the cGMP-dependent protein kinase specifically inhibited the histone kinase activity and the self-phosphorylation of the purified cGMP-dependent protein kinase without affecting the cGMP binding site. This antiserum also specifically inhibited the phosphorylation of the endogenous substrate proteins by endogenous cGMP-dependent protein kinase in smooth muscle membranes, but did not cross-react detectably with catalytic subunit or regulatory subunit of type I or type II cAMP-dependent protein kinase. Conversely, anti-sera against the regulatory subunit of type I or type II cAMP-dependent protein kinase did not cross-react detectably with cGMP-dependent protein kinase. The substantial differences between the immunological properties of the cGMP-dependent and cAMP-dependent protein kinases suggest that these two enzymes have distinct physiological roles.

Homogenates, membranes and cytosol of rat and human platelets were found to contain cGMP-dependent protein kinase immunoreactivity. Specific cGMP-dependent protein kinase immunoreactivity was about 1.7 pmol protein kinase/mg protein for homogenates of human platelets and 0.7 pmol/mg for homogenates of rat platelets; the majority appeared to be associated with the membrane fraction. In membranes of platelets low concentrations of cAMP (0.5-2 microM) stimulated the phosphorylation of five major proteins with apparent relative molecular masses, Mr, of 240 000, 130 000, 50 000, 42 000 and 22 000 while low concentrations of cGMP (0.5-2 microM) stimulated the phosphorylation of three major proteins with apparent Mr of 130 000, 50 000 and 46 000. An affinity-purified antibody against the cGMP-dependent protein kinase was prepared which specifically inhibited the activity of cGMP-dependent protein kinase. In membranes of human platelets this affinity-purified antibody inhibited the cGMP-stimulated phosphorylation of the three proteins with Mr of 130 000, 50 000 and 46 000 while it had no effect on the cAMP-dependent and cyclic-nucleotide-independent protein phosphorylation. The results demonstrate that platelets contain a cGMP-dependent protein kinase and at least three specific substrates for this enzyme. Two of these substrates, the proteins with apparent molecular Mr of 130 000 and 50 000, are substrates for both cAMP- and cGMP-dependent protein kinase. The protein with apparent Mr of 130 000 appears to be closely related to an intrinsic plasma membrane protein of vascular smooth muscle cells which is a substrate for a membrane-associated cGMP-dependent protein kinase. Therefore, cGMP-dependent protein kinase and cGMP-regulated phosphoproteins may mediate in platelets the intracellular effects of those hormones, vasodilators and drugs which elevate the level of cGMP and inhibit platelet aggregation.

The nonapeptide, bradykinin, elevated the level of cyclic GMP in two neural cell lines, neuroblastoma X glioma hybrid cells (clone 108CC15) and glioma cells (clone C6-4-2). In the hybrid cells the half-maximal stimulation occurred at 0.1 nM and the maximum was reached at 10 nM bradykinin. As soon as 30 s after the addition of bradykinin to the cultured cells, the intracellular concentration of cyclic GMP had increased maximally. The subsequent decline to the original level proceeded more slowly and lasted around 10 min. Hybrid cells incubated for 10 min in the presence of bradykinin and washed thereafter, did not respond at all to a subsequent 1 min challenge incubation with bradykinin. This nearly complete desensitization lasted for a period of 20 min. One hour after removal of bradykinin the original response to the peptide was restored. Modified and partial sequences of bradykinin were also investigated for their ability to induce the cyclic GMP response in the hybrid cells. Removal of amino acids from either terminus of bradykinin led to an almost complete loss of activity. The data are discussed with respect to our previous observation that bradykinin causes a slow hyperpolarization response in these cell lines and that on prolonged exposure to the peptide the membrane potential response of the cells is lost due to desensitization.

The mechanism of vasodilator-induced inhibition of platelet aggregation was investigated in human platelets. Cyclic nucleotide-elevating vasodilators stimulated cAMP- or cGMP-dependent protein phosphorylation, inhibited the activation of both protein kinase C and myosin light chain kinase, and inhibited the thrombin-induced hydrolysis of phosphatidylinositol-4,5-bisphosphate without affecting its resynthesis. The results suggest that cAMP- and cGMP-elevating vasodilators both inhibit platelet aggregation at an early step of the activation cascade, presumably at the level of phospholipase C.

Vasodilators such as sodium nitroprusside, nitroglycerin and various prostaglandins are capable of inhibiting platelet aggregation associated with an increase of either cGMP or cAMP. In our studies with intact platelets, prostaglandin E1 and sodium nitroprusside stimulated the phosphorylation of several proteins which could be distinguished from proteins known to be phosphorylated by a calmodulin-regulated protein kinase or by protein kinase C. Prostaglandin E1 (10 microM) or dibutyryl cAMP (2 mM) stimulated the phosphorylation of proteins with apparent relative molecular masses, Mr, of 240,000, 68,000, 50,000, and 22,000 in intact platelets. These proteins were also phosphorylated in response to low concentrations (1-2 microM) of cAMP in a particulate fraction of platelets. In intact platelets, sodium nitroprusside (100 microM) and the 8-bromo derivative of cGMP (2 mM) increased the phosphorylation of one protein of Mr 50,000 which was also phosphorylated in response to low concentrations (1-2 microM) of cGMP in platelet membranes. An additional protein (Mr 24,000) appeared to be phosphorylated to a lesser degree in intact platelets by prostaglandin E1 and sodium nitroprusside. Since the phosphorylation of the protein of Mr 50,000 was stimulated both in intact platelets by cyclic-nucleotide-elevating agents and cyclic nucleotide analogs, as well as in platelet membranes by cyclic nucleotides, this phosphoprotein was analyzed by limited proteolysis, tryptic fingerprinting and phosphoamino acid analysis. These experiments indicated that the 50-kDa proteins phosphorylated by sodium nitroprusside and prostaglandin E1 were identical, and that the peptide of the 50-kDa protein phosphorylated by both agents was also the same as the peptide derived from the 50-kDa protein phosphorylated in platelet membranes by cGMP- and cAMP-dependent protein kinases, respectively. Regulation of protein phosphorylation mediated by cAMP- and cGMP-dependent protein kinases may be the molecular mechanism by which those vasodilators, capable of increasing either cAMP or cGMP, inhibit platelet aggregation.