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Biomedical subjects

M Eigenthaler

Publications and source records attributed to M Eigenthaler.

At least 19 recordsLinked to original sources
PubMed2003

Prolactin receptor signaling during platelet activation.

Prolactin is a newly recognized platelet coactivator that functions through potentiation of ADP-induced platelet activation. However, the possible association between hyperprolactinemia and venous thromboembolism (VTE) has not been systematically investigated up to now; prolactin signaling mechanisms in platelets still need to be elucidated. In this study, plasma prolactin levels in healthy subjects and patients with VTE were determined, demonstrating that patients with VTE and no other congenital risk factors had significantly increased plasma prolactin levels. Moreover, prolactinoma patients demonstrated a higher incidence of VTE than the general population. To elucidate the molecular mechanisms for the development of venous thrombosis, prolactin receptor signaling during platelet activation was investigated with a focus on ADP-stimulated G-protein-regulated signaling pathways. The short isoform of prolactin receptors was detected on platelets. Signaling through this receptor, although not directly linked to Gq-proteins, substitutes for Gq-protein regulated signaling pathways involved in platelet activation. We identified protein kinase C, a well-established signaling molecule in platelet activation, as a target molecule for prolactin signaling pathways in human platelets. Our findings indicate that hyperprolactinemia may be an important novel risk factor for VTE, suggesting that its thrombogenic effect may be mediated through enhanced platelet reactivity. Revealing the molecular mechanisms of prolactin signaling will allow the design of new antithrombotic therapies.

Adult↗
PubMed2001

Taming platelets with cyclic nucleotides.

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.

Adenylyl Cyclases↗
PubMed2001

PRL as a novel potent cofactor for platelet aggregation.

Pregnancy (including puerperium) is a period of hypercoagulability and seems to be an independent major risk factor for venous thromboembolism (VTE). However, the basis of the increased risk of VTE in pregnancy and around delivery is unknown. We hypothesized that changes in PRL, which is a prominently increased hormone during pregnancy and lactation, might be involved in the activation of platelets. To investigate platelet functional abnormalities in pregnancy, we assessed the ADP-stimulated and nonstimulated P-selectin expression of platelets in 42 consecutive pregnant women, 22 normo- and hyperprolactinemic patients with pituitary tumors, and controls. In addition, the aggregation of platelets by human PRL in vitro was studied. We found a significant correlation between PRL values and ADP stimulation of platelets in pregnant women (r = 0.56; P < 0.0001) and patients with pituitary tumors (r = 0.57; P = 0.006). Hyperprolactinemic pregnant women or hyperprolactinemic patients with pituitary tumors revealed significantly higher ADP stimulation of platelets (P < 0.0001) than healthy controls or normoprolactinemic patients with pituitary tumors. These results were reconciled by increased in vitro stimulation and aggregation of platelets using human PRL. Our novel findings demonstrate that hyperprolactinemia causes increased platelet aggregation via ADP stimulation both in vitro and in vivo. Moreover, our data indicate that PRL may be a physiological cofactor of the delicate coagulation balance during pregnancy and puerperium that might explain the increased risk of VTE in pregnant women around delivery. Further studies of the interaction between PRL and platelets will clarify the clinical relevance of hyperprolactinemia as a potential risk factor for VTE.

Adenoma↗
PubMed2000

Inhibition of agonist-induced p42 and p38 mitogen-activated protein kinase phosphorylation and CD40 ligand/P-selectin expression by cyclic nucleotide-regulated pathways in human platelets.

Platelet activation and adhesion to endothelial cells and extracellular matrix proteins are crucial events in the development of arterial cardiovascular diseases. Platelet activation is initiated by stimulation of intracellular signaling cascades, including the p42 mitogen-activated protein kinase (MAPK) and p38 MAPK pathways, followed by major changes in the platelet cytoskeleton and expression and activation of platelet surface receptors, such as P-selectin (CD62P) and CD40 ligand (CD40L). Activated platelets directly bind to vascular endothelial cells via CD40L/CD40 interactions and induce inflammatory reactions that initiate or aggravate atherosclerotic lesions. The aim of this study was to investigate effects of two known platelet inhibitors-the cAMP-elevating prostaglandin E(1) (PG-E(1)) and the cGMP-elevating sodium nitroprusside (SNP)-on platelet p42 MAPK and p38 MAPK activation as well as on surface expression of CD62P and CD40L. MAPK activation was analyzed by Western blot experiments using phosphorylation-specific antibodies, and surface CD40L and CD62P expression was determined by flow cytometry analysis. PG-E(1) and SNP strongly inhibited p42 and p38 MAPK phosphorylation as well as CD40L and CD62P expression in response to thrombin, a thromboxane A(2) analog, and ADP. These data indicate that adenosine and guanosine 3',5'-cyclic monophosphate-dependent protein kinases not only inhibit platelet pathways leading to activation and aggregation, but also those resulting in enhanced surface expression of protein ligands involved in inflammation. Expression of CD40L and CD62P was found to be independent of MAPK activation, since it was not inhibited by specific MAPK inhibitors. Inhibition of platelet-induced inflammatory responses including CD62P- and CD40L-mediated interaction of platelets with leukocytes and endothelial cells, respectively, is suggested to be an important component of the long-term vasoprotective effects of cyclic nucleotide-elevating prostaglandins and NO donors.

Alprostadil↗
PubMed1999

Megakaryocyte hyperplasia and enhanced agonist-induced platelet activation in vasodilator-stimulated phosphoprotein knockout mice.

Vasodilator-stimulated phosphoprotein (VASP), a substrate of cAMP- and cGMP-dependent protein kinases, is associated with focal adhesions, cell-cell contacts, microfilaments, and highly dynamic membrane regions. VASP, which is expressed in most cell types and in particularly high levels in human platelets, binds to profilin, zyxin, vinculin, F-actin, and the Listeria monocytogenes surface protein ActA. VASP is a member of the enabled (Ena)/VASP protein family and is thought to be involved in actin filament formation and integrin alphaIIbbeta3 inhibition in human platelets. To gain further insight into the in vivo function of this protein, VASP-deficient mice were generated by homologous recombination. VASP-/- mice demonstrated hyperplasia of megakaryocytes in bone marrow and spleen but exhibited no other macroscopic or microscopic abnormalities. Activation of platelets with thrombin induced a more than 2-fold higher surface expression of P-selectin and fibrinogen binding in VASP-deficient platelets in comparison to wild type. These data support the concept that VASP is a negative modulator of platelet and integrin alphaIIbbeta3 activation. Although the limited phenotypic differences between wild-type and VASP-/- mice suggested functional compensation of VASP by members of the Ena/VASP family, alterations in the expression levels of mammalian enabled (Mena) and Ena-VASP-like (Evl) protein were not detected. VASP-deficient mice may provide an interesting model system for diseases in which enhanced platelet activation plays a major role.

Animals↗
PubMed1999

Specific impairment of human platelet P2Y(AC) ADP receptor-mediated signaling by the antiplatelet drug clopidogrel.

Clopidogrel is an effective new antiplatelet agent useful for the treatment of ischemic cerebrovascular, cardiac, and peripheral arterial disease. However, the mechanism of clopidogrel action is not well understood, although it is known to inhibit ADP-evoked platelet aggregation. In the current study, the effect of clopidogrel on recently identified human platelet ADP receptors and their signaling pathways was investigated by using platelets from clopidogrel-treated subjects, 6 healthy volunteers (2 females and 4 males) who received 75 mg of clopidogrel daily for 7 days. Blood was taken and various platelet receptor signaling pathways were analyzed before treatment, after 7 days of medication, and 4 weeks after treatment had ceased. Platelet tests included the analysis of aggregation, rapid calcium influx, calcium mobilization from intracellular stores, adenylyl cyclase, and phosphorylation of vasodilator-stimulated phosphoprotein (VASP). The data indicate that clopidogrel does not affect those platelet ADP receptors coupled to cation influx (P2X1 ADP receptors) or calcium mobilization (P2Y1 ADP receptors). In contrast, clopidogrel treatment specifically impairs the ADP receptor coupled to G(i)/adenylyl cyclase (P2Y(AC) ADP receptors). Clopidogrel abolishes the inhibitory P2Y(AC) receptor-mediated ADP effects on prostaglandin E(1)-stimulated, cAMP-dependent phosphorylation of VASP without affecting epinephrine, thrombin, and thromboxane signaling. VASP phosphorylation is known to be closely correlated with the inhibition of platelet and fibrinogen receptor (glycoprotein IIb/IIIa) activation. Therefore, inhibition of the platelet P2Y(AC) ADP receptor and its intracellular signaling, including decreased VASP phosphorylation, is suggested as a molecular mechanism of clopidogrel action.

Adenosine Diphosphate↗
PubMed1999

Flow cytometry analysis of intracellular VASP phosphorylation for the assessment of activating and inhibitory signal transduction pathways in human platelets--definition and detection of ticlopidine/clopidogrel effects.

Increased platelet adhesion or aggregation are key events in the pathogenesis of cardiovascular diseases. Exact determination of the platelet activation state is essential to recognize, prevent, and treat cardiovascular complications due to platelet dysfunction. Initial phases of platelet activation and inhibition are characterized by phosphorylation of specific intracellular proteins. However, methodological problems often prevent analysis of platelet protein phosphorylation under clinical conditions. A novel flow cytometry-based method using a phosphorylation-specific antibody was developed for fast and easy quantification of the phosphorylation state of a specific intracellular platelet protein. This method was used to analyze various platelet receptors and their intracellular signaling which may be impaired in genetic or acquired disorders or altered due to therapeutic interventions. In a first clinical application, the inhibitory effects of ticlopidine and clopidogrel on the platelet P2YAC ADP receptor were monitored.

Adult↗
PubMed1998

Functional analysis of cGMP-dependent protein kinases I and II as mediators of NO/cGMP effects.

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.

Animals↗
PubMed1997

Affinity modulation of platelet integrin alphaIIbbeta3 by beta3-endonexin, a selective binding partner of the beta3 integrin cytoplasmic tail.

Platelet agonists increase the affinity state of integrin alphaIIbbeta3, a prerequisite for fibrinogen binding and platelet aggregation. This process may be triggered by a regulatory molecule(s) that binds to the integrin cytoplasmic tails, causing a structural change in the receptor. beta3-Endonexin is a novel 111-amino acid protein that binds selectively to the beta3 tail. Since beta3-endonexin is present in platelets, we asked whether it can affect alphaIIbbeta3 function. When beta3-endonexin was fused to green fluorescent protein (GFP) and transfected into CHO cells, it was found in both the cytoplasm and the nucleus and could be detected on Western blots of cell lysates. PAC1, a fibrinogen-mimetic mAb, was used to monitor alphaIIbbeta3 affinity state in transfected cells by flow cytometry. Cells transfected with GFP and alphaIIbbeta3 bound little or no PAC1. However, those transfected with GFP/beta3-endonexin and alphaIIbbeta3 bound PAC1 specifically in an energy-dependent fashion, and they underwent fibrinogen-dependent aggregation. GFP/beta3-endonexin did not affect levels of surface expression of alphaIIbbeta3 nor did it modulate the affinity of an alphaIIbbeta3 mutant that is defective in binding to beta3-endonexin. Affinity modulation of alphaIIbbeta3 by GFP/beta3-endonexin was inhibited by coexpression of either a monomeric beta3 cytoplasmic tail chimera or an activated form of H-Ras. These results demonstrate that beta3-endonexin can modulate the affinity state of alphaIIbbeta3 in a manner that is structurally specific and subject to metabolic regulation. By analogy, the adhesive function of platelets may be regulated by such protein-protein interactions at the level of the cytoplasmic tails of alphaIIbbeta3.

Animals↗
PubMed1997

A conserved sequence motif in the integrin beta3 cytoplasmic domain is required for its specific interaction with beta3-endonexin.

Integrin signaling is mediated by interaction of integrin cytoplasmic domains with intracellular signaling molecules. Recently, we identified a novel 111-amino acid polypeptide, termed beta3-endonexin, which interacts selectively with the integrin beta3 cytoplasmic domain. In the present study we conducted a systematic mutational analysis of both the integrin beta3 cytoplasmic domain and beta3-endonexin to map sites required for interaction. The interaction of the full-length beta3 integrin subunit with beta3-endonexin in vitro required the beta3 cytoplasmic domain. In a yeast two-hybrid system, both membrane-proximal and membrane-distal residues of the beta3 cytoplasmic domain were necessary for interaction with beta3-endonexin. In particular, the membrane-distal NITY motif at beta3 756-759 was critical for the interaction. Exchange of beta3 residues 756-759 (NITY) for the corresponding residues in beta1 (NPKY) endowed the beta1 cytoplasmic domain with the ability to interact with beta3-endonexin. Conversely, exchange of the NPKY motif at beta1 772-775 for the NITY motif in beta3 abolished interaction of this chimeric cytoplasmic domain with beta3-endonexin. Because the NITY motif is present in the beta3 but not the beta1 cytoplasmic domain, these results explain the selective interaction of this cytoplasmic domain with beta3-endonexin. In addition, deletional analysis suggested that a core 91-residue sequence of beta3-endonexin is sufficient for specific binding to the beta3 cytoplasmic domain. These studies have identified a cytoplasmic domain sequence motif that specifies an integrin-specific protein-protein interaction.

Amino Acid Sequence↗
PubMed1996

Distribution and activation of cAMP- and cGMP-dependent protein kinases in highly purified human platelet plasma and intracellular membranes.

Previously cAMP- and cGMP-dependent protein kinases (cAMP-PK, cGMP-PK) have been found predominantly associated with the particulate fraction in human platelets. We now report the distribution and activation of cAMP-PK and cGMP-PK in highly purified fractions of human platelet plasma (PM) and intracellular membranes (IM) prepared using high voltage free flow electrophoresis. Two non-hydrolysable analogues of cAMP and cGMP namely Sp-5,6-DCI-cBiMPS and 8-p-CPT-cGMP have been used to activate cAMP-PK and cGMP-PK respectively. Addition of either agonist with [gamma 32P]ATP stimulated the endogenous activity of cAMP-PK or cGMP-PK in PM but not in IM. With PM Sp-5,6-DCI-cBiMPS stimulated the phosphorylation of protein substrates of Mr 16, 22, 24, 46-50, 66, 90, 160 and 250 kDa. A specific peptide inhibitor of cAMP-PK inhibited the phosphorylation of all of the substrates by Sp-5,6-DCI-cBiMPS. 8-pCPT-cGMP also induced the phosphorylation of a number of substrates particularly 16, 22, 46-50, 90 and 250 kDa proteins. Inclusion of the cAMP-PK inhibitor peptide totally blocked the phosphorylation of the 16 and 22 kDa proteins, partially inhibited phosphorylation of 46-50 and 90 kDa proteins and had no effect on the 250 kDa protein indicating the 46-50, 90 and 250 kDa proteins were also cGMP-PK substrates. Western blotting with antibodies to cGMP-PK and the catalytic subunit of cAMP-PK revealed the presence of the kinases to be exclusively associated with PM with no detection in IM. The presence of cAMP-PK substrates in IM was investigated by exogenous addition of catalytic subunit of cAMP-PK. Phosphoproteins of Mr 16, 22, 27, 30, 45, 75, 116 and 250 kDa were detected. A range of antibodies to cAMP-PK substrates were used to identify and localise the substrates. These antibodies revealed GPIb and VASP to be exclusively associated with PM fractions. Rap IB was also predominantly associated with PM with a small level detected in IM. Antibodies to the IP3 receptor (18A 10 and 4C11) revealed the protein to be predominantly associated with IM. Additionally the antibody 4C11 recognised a 230 kDa protein band in PM that was not seen in IM. From the known specificity of these antibodies the results confirm the presence of a type 1 IP3 receptor in IM and a distinct (possible type III) IP3 receptor with the PM. The 16, 22, 27, 30, 75 and 116 kDa proteins in IM represent newly detected substrates for cAMP-PK of presently unknown identity.

Blood Platelets↗
PubMed1995

Beta 3-endonexin, a novel polypeptide that interacts specifically with the cytoplasmic tail of the integrin beta 3 subunit.

The adhesive and signaling functions of integrins are regulated through their cytoplasmic domains. We identified a novel 111 residue polypeptide, designated beta 3-endonexin, that interacted with the cytoplasmic tail of the beta 3 integrin subunit in a yeast two-hybrid system. This interaction is structurally specific, since it was reduced by 64% by a point mutation in the beta 3 cytoplasmic tail (S752-->P) that disrupts integrin signaling. Moreover, this interaction is integrin subunit specific since it was not observed with the cytoplasmic tails of the alpha IIb, beta 1, or beta 2 subunits. beta 3-Endonexin fusion proteins bound selectively to detergent-solubilized beta 3 from platelets and human umbilical vein endothelial cells, and beta 3-endonexin mRNA and protein were detected in platelets and other tissues. A related mRNA encoded a larger polypeptide that failed to bind to beta integrin tails. The apparent specificity of beta 3-endonexin for the beta 3 integrin subunit suggests potential mechanisms for selective modulation of integrin functions.

Amino Acid Sequence↗
PubMed1994

Synergistic phosphorylation of the focal adhesion-associated vasodilator-stimulated phosphoprotein in intact human platelets in response to cGMP- and cAMP-elevating platelet inhibitors.

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.

Alprostadil↗
PubMed1994

(Rp)-8-pCPT-cGMPS, a novel cGMP-dependent protein kinase inhibitor.

In the present study, the inhibitory effect of the cGMP analog (Rp)-8-(para-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate ((Rp)-8-pCPT-cGMPS) on the cGMP-dependent protein kinase-mediated protein phosphorylation in intact human platelets was investigated. In vitro phosphorylation experiments with the substrate kemptide demonstrated an inhibition of the cGMP-dependent protein kinase by (Rp)-8-pCPT-cGMPS with a Ki of 0.5 microM. In intact human platelets, (Rp)-8-pCPT-cGMPS antagonized the activation of the cGMP-dependent protein kinase by 8-pCPT-cGMP without affecting cAMP-dependent protein kinase or cGMP-regulated phosphodiesterases. The data obtained suggest that (Rp)-8-pCPT-cGMPS may be a useful tool for studying the role of cGMP in vitro and in intact cells.

Animals↗
PubMed1994

Endothelium-dependent phosphorylation of vasodilator-stimulated protein in platelets during coronary passage.

Compounds that elevate intraplatelet guanosine 3',5'-cyclic monophosphate (cGMP) or adenosine 3',5'-cyclic monophosphate (cAMP) stimulate the phosphorylation of a 46- to 56-kDa thrombocyte protein designated "vasodilator-stimulated protein" (VASP), which is most likely involved in the regulation of adhesion/aggregation. We investigated whether endothelium-derived relaxing factor (EDRF)/nitric oxide (NO) and prostaglandin I2 (PGI2) affected VASP phosphorylation in washed human platelets that were injected into the coronaries of saline-perfused rabbit hearts (n = 22) and collected immediately after passage. The endothelial stimulator acetylcholine (ACh; 1 microM) significantly increased the concentration of cGMP (indicating release of EDRF) and PGI2 in the coronary venous effluent, as well as the concentration of cGMP and cAMP in platelets. Phosphorylation state of VASP increased from 32.1 +/- 2.9 to 64.8 +/- 2.7%. Inhibition of EDRF/NO synthesis by NG-nitro-L-arginine (30 microM) completely abolished the ACh-induced cGMP increase, attenuated the cAMP-elevation without affecting PGI2, and caused a 20.5 +/- 5.8% decrease of the phosphorylation state of VASP. Indomethacin (30 microM) virtually abolished ACh-induced increases of PGI2, cAMP (but not cGMP), and phosphorylated VASP. These results indicate that both EDRF/NO and PGI2 contribute to the regulation of VASP phosphorylation in platelets collected after a single coronary passage. Their synergistic inhibitory effects on platelet function may thus be mediated by a common effect on target proteins like VASP as well as by a secondary increase in cAMP in response to cGMP-elevating compounds such as EDRF.

Acetylcholine↗
PubMed1993

Defective nitrovasodilator-stimulated protein phosphorylation and calcium regulation in cGMP-dependent protein kinase-deficient human platelets of chronic myelocytic leukemia.

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.

Blood Platelets↗
PubMed1993

Role of cyclic nucleotide-dependent protein kinases and their common substrate VASP in the regulation of 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.

Adenylyl Cyclases↗