Vasopressin acts on platelets to generate procoagulant activity

The aim of our study was to examine whether arginine vasopressin (AVP) is able to evoke in human platelets a procoagulant response due to activation of an Na+/H+ exchanger. It was found that treatment of platelets with AVP (20-100 nmol/l) results in generation of a weak calcium signal, activation of Na+/H+ exchanger, aggregation, and development of a procoagulant response. The AVP-evoked procoagulant response was dose and time dependent, weaker than that produced by collagen or monensin (mimics Na+/H+ exchanger), and less pronounced following the inhibition of Na+/H+ exchanger by 5-(N-ethyl-N-isopropyl) amiloride or genistein. Flow cytometry studies reveal that in-vitro platelet treatment with AVP results in an unimodal left shift in the forward and side scatter of the entire platelet population, indicating morphological changes on the plasma membrane. The shift was dose related, weaker than that evoked by collagen, similar to that produced by monensin and strongly reduced in the presence of 5-(N-ethyl-N-isopropyl) amiloride or genistein. Using flow cytometry, we demonstrated enhanced expression of phosphatidylserine on the AVP-treated platelets. AVP-evoked phosphatidylserine exposure was dose dependent, inhibited by 5-(N-ethyl-N-isopropyl) amiloride or genistein and weaker than that produced by collagen. AVP in a dose-dependent manner produced a rise in platelet volume. The swelling was inhibited by 5-(N-ethyl-N-isopropyl) amiloride, and its kinetics was similar to that observed in the presence of monensin. We conclude that prolonged treatment of platelets with AVP results in a procoagulant response, which may occur as a consequence of Na+ influx mediated by Na+/H+ exchanger.     

Inhibition of Na+/Ca2+ exchange in membrane vesicle and papillary muscle preparations from guinea pig heart by analogs of amiloride.

Na+/Ca2+ exchange is inhibited in both guinea pig cardiac membrane vesicles and papillary muscles in a concentration-dependent fashion by several analogs of the pyrazine diuretic amiloride. Structure/activity studies based on transport measurements in vesicles prepared from guinea pig left ventricle indicate that hydrophobic substitutions at the terminal nitrogen atom of the guanidinium moiety of amiloride improved the inhibitory potency almost 100-fold over that of the parent compound. 3',4'- Dichlorobenzamil ( DCB ) is one of the most active inhibitors (IC50 = 17 microM). In electrically stimulated papillary muscles isolated from guinea pig heart, 10-40 microM DCB decreases contractile force. At 100 microM inhibitor, diastolic tension is significantly increased. The positive inotropic responses to veratridine and ouabain are inhibited by 20 and 40 microM DCB . Since the responses to these interventions were a consequence of increased intracellular Na+ concentration, these data indicate that DCB is an inhibitor of Na+-dependent Ca2+ influx in the intact tissue. Interpretation of mechanical responses elicited by paired pulses suggests that 40 microM but not 100 microM DCB decreases release of Ca2+ from the sarcoplasmic reticulum. The mechanical data obtained with concentrations of DCB that inhibited Na+/Ca2+ exchange in vesicles suggest that a significant amount of Ca2+ can enter the cardiac cell via Na+/Ca2+ exchange under normal conditions and that this transport system may be an important source of Ca2+ supplying the sarcoplasmic reticulum in guinea pig heart. Moreover, these amiloride analogs function as potent inhibitors of the positive inotropic effect caused by increased intracellular Na+ concentration.     

Inhibitory effects of amiloride and its analogues on prostaglandin E2-stimulated fluid transport by cultured porcine thyroid cells: evidence for apical membrane Na+ channels

Confluent monolayers of cultured porcine thyroid cells transport fluid from the apical to the basal surface, forming circumscribed zones of detachment (domes) from the culture dish substrate. Fluid transport, as measured by increase in dome height, was stimulated by prostaglandin E2 (PGE2; 1 mumol/l) and inhibited by amiloride (0.1-100 mumol/l). Values of the inhibition constant (Ki) with 95% confidence limits for each of a series of amiloride analogues were: 3',4'-dichlorobenzamil (DCB), 0.090 (0.045-0.18) mumol/l; 2',4'-dimethylbenzamil (DMB), 0.14 (0.074-0.27) mumol/l; amiloride, 0.72 (0.33-1.8) mumol/l; 5-(N,N-hexamethylene)amiloride (HMA), 17 (5.9-43) mumol/l; 5-(N-ethyl-N-isopropyl)amiloride (EIPA), 33 (15-71) mumol/l; and 2-guanidinobenzimidazole, 243 (110-570) mumol/l. Triaminopyrimidine was ineffective at concentrations up to 1 mmol/l. Since DCB and DMB are known to have a higher affinity for Na+/H+ channels, while HMA and EIPA show higher affinity for Na+/H+ antiports, it was concluded that PGE2-stimulated fluid transport involved an apical membrane Na+ channel.     

Differential requirements for platelet aggregation and inhibition of adenylate cyclase by epinephrine. Studies of a familial platelet alpha 2-adrenergic receptor defect

We describe a family whose members have impaired platelet aggregation and secretion responses to epinephrine with normal responses to adenosine diphosphate and collagen. Platelet alpha 2-adrenergic receptors (measured using 3H methyl-yohimbine) were diminished in the propositus (78 sites per platelet), his two sisters (70 and 27 sites per platelet), and parents (37 and 63 sites per platelet), but not in two maternal aunts (12 normal subjects, 214 +/- 18 sites per platelet; mean +/- SE). However, the inhibition of cyclic adenosine monophosphate (cAMP) levels by epinephrine in platelets exposed to 400 nmol/L PGI2 was similar in the patients and five normal subjects (epinephrine concentration for 50% inhibition, 0.04 +/- 0.01 mumol/L v 0.03 +/- 0.01 mumol/L; P greater than .05). In normal platelets, the concentration of yohimbine (0.18 mumol/L) required for half maximal inhibition of aggregation induced by 2 mumol/L epinephrine was lower than that for inhibition of its effect on adenylate cyclase (1.6 mumol/L). In quin2 loaded platelets, thrombin (0.1 U/mL) stimulated rise in cytoplasmic Ca2+ concentration, [Ca2+]i, was normal in the two patients studied. The PGI2 analog ZK 36,374 completely inhibited thrombin-induced rise in [Ca2+]i; the reversal of this inhibition by epinephrine was normal in the two patients. Thus, despite the impaired aggregation response to epinephrine, platelets from these patients have normal ability to inhibit PGI2-stimulated cAMP levels. These patients with an inherited receptor defect provide evidence that fewer platelet alpha 2-adrenergic receptors are required for epinephrine-induced inhibition of adenylate cyclase than for aggregation.     

Fibrinogen binding to the integrin alpha(IIb)beta(3) modulates store-mediated calcium entry in human platelets

Effects of the occupation of integrin alpha(IIb)beta(3) by fibrinogen on Ca(++) signaling in fura-2-loaded human platelets were investigated. Adding fibrinogen to washed platelet suspensions inhibited increases in cytosolic [Ca(++)] concentrations ([Ca(++)](i)) evoked by adenosine diphosphate (ADP) and thrombin in a concentration-dependent manner in the presence of external Ca(++) but not in the absence of external Ca(++) or in the presence of the nonselective cation channel blocker SKF96365, indicating selective inhibition of Ca(++) entry. Fibrinogen also inhibited store-mediated Ca(++) entry (SMCE) activated after Ca(++) store depletion using thapsigargin. The inhibitory effect of fibrinogen was reversed if fibrinogen binding to alpha(IIb)beta(3) was blocked using RDGS or abciximab and was absent in platelets from patients homozygous for Glanzmann thrombasthenia. Fibrinogen was without effect on SMCE once activated. Activation of SMCE in platelets occurs through conformational coupling between the intracellular stores and the plasma membrane and requires remodeling of the actin cytoskeleton. Fibrinogen inhibited actin polymerization evoked by ADP or thapsigargin in control cells and in cells loaded with the Ca(++) chelator dimethyl BAPTA. It also inhibited the translocation of the tyrosine kinase p60(src) to the cytoskeleton. These results indicate that the binding of fibrinogen to integrin alpha(IIb)beta(3) inhibits the activation of SMCE in platelets by a mechanism that may involve modulation of the reorganization of the actin cytoskeleton and the cytoskeletal association of p60(src). This action may be important in intrinsic negative feedback to prevent the further activation of platelets subjected.     

Recombinant factor VIIa enhances deposition of platelets with congenital or acquired alpha IIb beta 3 deficiency to endothelial cell matrix and collagen under conditions of flow via tissue factor-independent thrombin generation

A novel approach to treat bleeding episodes in patients with Glanzmann thrombasthenia (GT) and perhaps also in patients receiving alpha IIb beta 3 inhibitors is the administration of recombinant factor VIIa (rFVIIa). The mechanism of action of rFVIIa in these patients is, however, still unclear. We studied the effect of rFVIIa-mediated thrombin formation on adhesion of alpha IIb beta 3-deficient platelets under flow conditions. Adhesion of alpha IIb beta 3-deficient platelets to the extracellular matrix (ECM) of stimulated human umbilical vein endothelial cells or to collagen type III was studied using a model system with washed platelets and red cells. When alpha IIb beta 3-deficient platelets were perfused over the surface at arterial shear rate for 5 minutes, a low surface coverage was observed (GT platelets, mean +/- SEM, 37.5% +/- 5.0%; normal platelets preincubated with an RGD-containing peptide, 7.4% +/- 2.1%). When rFVIIa, together with factors X and II, was added to the perfusate, platelet deposition significantly increased (GT platelets, mean +/- SEM, 67.0% +/- 4.3%; normal platelets preincubated with an RGD-containing peptide, 48.2% +/- 2.9%). The same effect was observed when normal platelets were pretreated with the commercially available anti-alpha IIb beta 3 drugs abciximab, eptifibatide, or tirofiban. It was shown that tissue factor-independent thrombin generation (presumably induced by binding of rFVIIa to adhered platelets) was responsible for the increase in platelet deposition. In conclusion, defective adhesion of alpha IIb beta 3-deficient platelets to ECM can be restored by tissue factor-independent rFVIIa-mediated thrombin formation. The enhanced generation of platelet procoagulant surface facilitates fibrin formation, so that lack of platelet aggregate formation might be compensated for.     

Activation of the fibrinogen binding site on platelets isolated from a patient with the Strasbourg I variant of Glanzmann's thrombasthenia

One proposed ligand binding site on platelet integrin alpha IIb beta 3 is the region of the beta 3 subunit encompassing amino acids 211-221. However, we recently showed that synthetic peptides corresponding to amino acids 211-221 inhibit fibrinogen binding to alpha IIb beta 3 by binding to alpha IIb beta 3 and not to fibrinogen. In this study, we show that AP6, a monoclonal antibody (MoAb) directed against amino acids 214-221 of beta 3, bound to immobilized active alpha IIb beta 3 but did not inhibit fibrinogen binding to the complex. We then determined whether nonfunctional alpha IIb beta 3 on platelets with a beta 3 Arg-214-->Trp mutation (Strasbourg I variant of Glanzmann's thrombasthenia or GTV) could be induced to aggregate after treatment with dithiothreitol (DTT). DTT has been shown to expose the fibrinogen receptor on normal platelets. DTT treatment of GTV platelets did result in the formation of the fibrinogen binding site as indicated by the binding of pI-55, an MoAb that only binds to the activated form of alpha IIb beta 3. Furthermore, DTT-treated GTV platelets aggregated in the presence of fibrinogen and divalent cations. This aggregation was inhibited by EDTA, RGDS, and the selective alpha IIb beta 3 antagonist, Ro 43-5054. These data show that Arg-214 of beta 3 is not required for fibrinogen binding or for platelet aggregation. However, this amino acid appears to be critical for the formation and for the maintenance of the correct tertiary structure of the fibrinogen binding site on alpha IIb beta 3.     

Inhibition of platelet integrin alpha(IIb)beta(3) by peptides that interfere with protein kinases and the beta(3) tail

alpha-Thrombin stimulation of human platelets initiates inside-out signaling to integrin alpha(IIb)beta(3) (glycoprotein IIb/IIIa), resulting in the exposure of ligand binding sites. In the present study, the regulation of alpha(IIb)beta(3) via protein kinases was investigated in platelets permeabilized with streptolysin O by introducing peptides that interfere with these enzymes and with possible regulatory domains in the cytosolic tail of the beta(3) subunit. Compared with intact platelets, the permeabilized platelets preserved >80% of the aggregation, secretion, and alpha(IIb)beta(3) ligand binding capacity. The peptide YIYGSFK, a substrate for Src kinases, inhibited alpha-thrombin-induced ligand binding to alpha(IIb)beta(3), but a reversed peptide with Y-->F substitutions (KFSGFIF) had no effect. Ligand binding to alpha(IIb)beta(3) was also inhibited by the peptide RKRCLRRL, which binds irreversibly to the catalytic domain of protein kinase C. Peptides corresponding to parts of the protein C inhibitor and beta(2)-glycoprotein I were used as negative controls and failed to interfere with ligand binding. Possible target domains for protein kinases are present in the cytoplasmic tail of the beta(3) subunit. The LLITIHDR peptide, matching the membrane-proximal domain of beta(3) (residues 717 to 724), had no effect, but NNPLYKEA (residues 743 to 750), EATSTFTN (residues 749 to 756), and TNITYRGT (residues 755 to 762), which mimicked overlapping domains of the carboxy-terminal part of beta(3), reduced alpha-thrombin-induced ligand binding by 60+/-4%, 97+/-1%, and 97+/-2% (n=3) at 500 micromol/L peptide, respectively. These observations indicate that Src kinases and protein kinase C take part in inside-out signaling to integrin alpha(IIb)beta(3) and identify target domains in beta(3) that contribute to the regulation of this integrin.     

Sequence 274-368 in the beta 3-subunit of the integrin alpha IIb beta 3 provides a ligand recognition and binding domain for the gamma-chain of fibrinogen that is independent of platelet activation

Several bacterial-expressed recombinant fragments encompassing the extracellular part of the beta 3 subunit of the integrin alpha IIb beta 3 were shown to recognize and bind soluble and immobilized forms of fibrinogen. Two of them, designated as rIII-11 (beta 3 274-368) and rIII-13 (beta 3 274-403), did not contain the established RGD-ligand binding sequence. In fact, they interacted, in a Ca(2+)-independent manner, with the C-terminal part of the fibrinogen gamma chain. Both beta 3 fragments blocked the participation of fibrinogen in the induction of platelet aggregation induced by adenosine diphosphate. Fragment rIII-13 was recognized by the anti-beta 3 monoclonal antibody B2A. This antibody, which possesses an epitope exposed on both resting and activated platelets, inhibited fibrinogen binding as well as platelet adhesion and aggregation. In conclusion, the results demonstrated that the 274-368 sequence of the beta 3 subunit of integrin alpha IIb beta 3 constitutes a fibrinogen ligand binding domain, distinct from the RGD-binding site, that is required for both platelet adhesion and aggregation.     

Regulation of c-jun-NH2 terminal kinase and extracellular-signal regulated kinase in human platelets

Platelets are an interesting model for studying the relationship betwen adhesion and mitogen-activated protein (MAP) kinase activation. We have recently shown that in platelets, ERK2 was activated by thrombin and downregulated by alpha(IIb)beta(3) integrin engagement. Here we focused our attention on the c-Jun NH2-terminal kinases (JNKs) and their activation in conditions of platelet aggregation. We found that JNK1 was present in human platelets and was activated after thrombin induction. JNK1 phosphorylation was detected with low concentrations of thrombin (0. 02 U/mL) and after 1 minute of thrombin-induced platelet aggregation. JNK1 activation was increased (fivefold) when fibrinogen binding to alpha(IIb)beta(3) integrin was inhibited by the Arg-Gly-Asp-Ser (RGDS) peptide or (Fab')(2) fragments of a monoclonal antibody specific for alpha(IIb)beta(3), demonstrating that, like ERK2, alpha(IIb)beta(3) integrin engagement negatively regulates JNK1 activation. Comparison of JNK1 activation by thrombin in stirred and unstirred platelets in the presence of RGDS peptide showed a positive regulation by stirring itself, independently of alpha(IIb)beta(3) integrin engagement, which was confirmed in a thrombasthenic patient lacking platelet alpha(IIb)beta(3). The same positive regulation by stirring was found for ERK2. These results suggest that MAP kinases (JNK1 and ERK2) are activated positively by thrombin and stirring. In conclusion, we found that JNK1 is present in platelets and can be activated after thrombin induction. Moreover, this is the first report showing that two different MAP kinases (ERK2 and JNK1) are regulated negatively by alpha(IIb)beta(3) engagement and positively by mechanical forces in platelets.     

Integrin alpha IIb beta 3 mediates binding of the Lyme disease agent Borrelia burgdorferi to human platelets.

Lyme disease is a chronic, multisystemic infection caused by the tick-borne spirochete Borrelia burgdorferi. Attachment of the spirochete to host cells via specific receptors is likely to be important in the establishment of infection. B. burgdorferi have previously been shown to bind to a variety of mammalian cells in vitro. Here we demonstrate that binding of B. burgdorferi to human platelets is mediated by the integrin alpha IIb beta 3 (glycoprotein IIb-IIIa), a critical receptor in thrombosis and hemostasis. Functional expression of this receptor requires platelet activation, and binding of the spirochete was observed only to activated platelets. Binding was inhibited by a synthetic Arg-Gly-Asp peptide that blocks ligand interaction with many integrins and by a synthetic peptide based on the gamma chain of fibrinogen that blocks binding to alpha IIb beta 3. In addition, attachment of the spirochete to platelets was inhibited by monoclonal antibodies directed against alpha IIb beta 3 that are known to block ligand-receptor interaction. No inhibition was seen with control peptides or with antibodies directed against other platelet receptors. B. burgdorferi bound efficiently to purified alpha IIb beta 3 but did not bind to platelets deficient in this integrin. Efficient platelet binding was displayed by a cloned, infectious B. burgdorferi strain, whereas a cloned noninfectious strain did not bind to platelets. Binding to integrins may be important for the ability of B. burgdorferi to establish infection in the diverse tissues affected by Lyme disease.     

Ser-752-->Pro mutation in the cytoplasmic domain of integrin beta 3 subunit and defective activation of platelet integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) in a variant of Glanzmann thrombasthenia.

Integrins are membrane receptors which mediate cell-cell or cell-matrix adhesion. Integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) acts as a fibrinogen receptor of platelets and mediates platelet aggregation. Platelet activation is required for alpha IIb beta 3 to shift from noncompetent to competent for binding soluble fibrinogen. The steps involved in this transition are poorly understood. We have studied a variant of Glanzmann thrombasthenia, a congenital bleeding disorder characterized by absence of platelet aggregation and fibrinogen binding. The patient's platelets did not bind fibrinogen after platelet activation by ADP or thrombin, though his platelets contained alpha IIb beta 3. However, isolated alpha IIb beta 3 was able to bind to an Arg-Gly-Asp-Ser affinity column, and binding of soluble fibrinogen to the patient's platelets could be triggered by modulators of alpha IIb beta 3 conformation such as the Arg-Gly-Asp-Ser peptide and alpha-chymotrypsin. These data suggested that a functional Arg-Gly-Asp binding site was present within alpha IIb beta 3 and that the patient's defect was not secondary to a blockade of alpha IIb beta 3 in a noncompetent conformational state. This was evocative of a defect in the coupling between platelet activation and alpha IIb beta 3 up-regulation. We therefore sequenced the cytoplasmic domain of beta 3, following polymerase chain reaction (PCR) on platelet RNA, and found a T-->C mutation at nucleotide 2259, corresponding to a Ser-752-->Pro substitution. This mutation is likely to be responsible for the uncoupling of alpha IIb beta 3 from cellular activation because (i) it is not a polymorphism, (ii) it is the only mutation in the entire alpha IIb beta 3 sequence, and (iii) genetic analysis of the family showed that absence of the Pro-752 beta 3 allele was associated with the normal phenotype. Our data thus identify the C-terminal portion of the cytoplasmic domain of beta 3 as an intrinsic element in the coupling between alpha IIb beta 3 and platelet activation.     

Sequential cytoplasmic calcium signals in a 2-stage platelet activation process induced by the glycoprotein Ibalpha mechanoreceptor

We found that the interaction of platelets with immobilized von Willebrand factor (VWF) under flow induces distinct elevations of cytosolic Ca(++) concentration ([Ca(++)](i)) that are associated with sequential stages of integrin alpha(IIb)beta(3) activation. Fluid-dynamic conditions that are compatible with the existence of tensile stress on the bonds between glycoprotein Ibalpha (GPIbalpha) and the VWF A1 domain led to Ca(++) release from intracellular stores (type alpha/beta peaks), which preceded stationary platelet adhesion. Raised levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate, as well as membrane-permeable calcium chelators, inhibited these [Ca(++)](i) oscillations and prevented stable adhesion without affecting the dynamic characteristics of the typical platelet translocation on VWF mediated by GPIbalpha. Once adhesion was established through the integrin alpha(IIb)beta(3), new [Ca(++)](i) oscillations (type gamma) of greater amplitude and duration, and involving a transmembrane ion flux, developed in association with the recruitment of additional platelets into aggregates. Degradation of released adenosine diphosphate (ADP) to AMP or inhibition of phosphatidylinositol 3-kinase (PI3-K) prevented this response without affecting stationary adhesion and blocked aggregation. These findings indicate that an initial signal induced by stressed GPIbalpha-VWF bonds leads to alpha(IIb)beta(3) activation sufficient to support localized platelet adhesion. Then, additional signals from ADP receptors and possibly ligand-occupied alpha(IIb)beta(3), with the contribution of a pathway involving PI3-K, amplify platelet activation to the level required for aggregation. Our conclusions modify those proposed by others regarding the mechanisms that regulate signaling between GPIbalpha and alpha(IIb)beta(3) and lead to platelet adhesion and aggregation on immobilized VWF.     

Na+ dependence of gonadotropin-releasing hormone action: characterization of the Na+/H+ antiport in pituitary gonadotropes.

GnRH stimulates LH release from gonadotropes in a Ca2(+)-dependent manner. Because of the apparent relationship between cellular Ca2+ metabolism and Na(+)-driven antiports, we investigated their influence on GnRH action. We also assessed the influence of bicarbonate, because its transport may alter effects of Na+/H+ exchange on intracellular pH. In pituitary cell cultures without bicarbonate, GnRH-stimulated LH release was reduced by Na+ omission, by amiloride, and by amiloride analogs that selectively block Na+/H+ exchange. The Na+ dependence of amiloride action (EC50, 14 and 100 microM in medium with 20 and 135 mM NaCl, respectively, and no effect in Na(+)-free medium) and the order of potency of these analogs, indicated specific inhibition of Na+/H+ exchange. 5-(N,N-Di-methyl)amiloride (DMA; a potent Na+/H+ exchange inhibitor) reduced GnRH-stimulated LH release but not GnRH receptor binding or Ca2+ ionophore (A23187)-stimulated LH release, suggesting inhibition at a locus beyond receptor occupancy but before exocytosis. Amiloride analogs that selectively inhibit Na+/Ca2+ exchange also modestly reduced GnRH-stimulated LH release. Bicarbonate (10 mM) reduced the inhibitory effects of DMA and Na+ omission (but not the effects of the Na+/Ca2+ exchange inhibitors or of a Ca2+ channel antagonist), and the effect of bicarbonate was inhibited by a blocker of bicarbonate-dependent antiports. These observations reveal the Na+ dependence of GnRH action and that gonadotropes possess a Na+/H+ exchanger. The Na+ dependence of GnRH-stimulated LH release appears to reflect at least in part dependence upon this antiport. Prevention of the Na+/H+ exchange inhibitor effects by bicarbonate supports the specificity of their action, but suggests regulation of this antiport as an unlikely means of controlling LH release in vivo.     

Factor H binds to platelet integrin alphaIIbbeta3

Factor H is a plasma protein that regulates activity of the alternative complement pathway in plasma and on cell surface. Binding of factor H to a cell surface protects that cell against complement-induced damage. Factor H binds to glycosoaminoglycans, surface-immobilized C3b, L selectin, and integrins such as alpha(M)beta(1) (a direct binding) or alpha(V)beta(3) (an indirect binding mediated through intermediary plasma proteins). We studied the binding of factor H to platelets and to integrin alpha(IIb)beta(3) (glycoprotein IIb-IIIa), the most abundant integrin on platelets. We measured binding of purified factor H to platelets or heterologous cells expressing recombinant alpha(IIb)beta(3) using flow cytometry. We also measured binding of factor H to alpha(IIb)beta(3) in cell free systems using either surface plasmon resonance or enzyme-linked immunosorbent assay. We found that factor H directly binds to alpha(IIb)beta(3) and this binding has a dissociation constant (Kd) of 131 +/- 60.9 nM and is not dependent on active conformation of alpha(IIb)beta(3) or on the presence of cations. Considering the high affinity of this interaction, the abundance of alpha(IIb)beta(3) integrin on platelets, and the high concentration of factor H in plasma, alpha(IIb)beta(3) provides a constitutive presence of factor H on platelets. Activation of platelets increases platelet-bound factor; however, this increase in binding of factor H cannot be explained by additional binding of factor H to alpha(IIb)beta(3) and perhaps involves other binding sites for factor H on platelets.     

Inhibitory mechanisms of kinetin,a plant growth-promoting hormone,in platelet aggregation

Kinetin has been shown to have anti-aging effects on several different systems including plants and human cells. The aim of this study was to examine the detailed inhibitory mechanisms of kinetin in platelet aggregation. In this study, kinetin concentration-dependently (50-150 microM) inhibited platelet aggregation in human platelets stimulated by agonists. Kinetin (70 and 150 microM) also concentration-dependently inhibited intracellular Ca2+ mobilization and phosphoinositide breakdown in platelets stimulated by collagen (1 microg/ml). Kinetin (70 and 150 microM) significantly inhibited thromboxane A2 formation stimulated by collagen (1 microg/ml) and arachidonic acid (60 microM) in human platelets. In addition, kinetin (70 and 150 microM) significantly increased the formation of cyclic AMP. Intracellular pH values were measured spectrofluorometrically using the fluorescent probe BCECF-AM in platelets. The thrombin-evoked increase in pHi was markedly inhibited in the presence of kinetin (70 and 150 microM). Rapid phosphorylation of a platelet protein of molecular weight (Mr) 47000 (P47), a marker of protein kinase C activation, was triggered by collagen (1 microg/ml). This phosphorylation was inhibited by kinetin (70 and 150 microM). In conclusion, these results indicate that the anti-platelet activity of kinetin may be involved in the following pathways: kinetin's effects may initially be due to inhibition of the activation of phospholipase C and the Na+/H+ exchanger. This leads to lower intracellular Ca2+ mobilization, followed by inhibition of TxA2 formation and then increased cyclic AMP formation, followed by a further inhibition of the Na+/H+ exchanger, ultimately resulting in markedly decreased intracellular Ca2+ mobilization and phosphorylation of P47. These results suggest that kinetin has an effective anti-platelet effect and that it may be a potential therapeutic agent for arterial thrombosis.     

The Ig-ITIM superfamily member PECAM-1 regulates the "outside-in" signaling properties of integrin alpha(IIb)beta3 in platelets

Previous studies have implicated the immunoglobulin (Ig)-immunoreceptor tyrosine-based inhibitory motif (ITIM) superfamily member platelet endothelial cell adhesion molecule-1 (PECAM-1) in the regulation of integrin function. While PECAM-1 has been demonstrated to play a role as an inhibitory coreceptor of immunoreceptor tyrosine-based activation motif (ITAM)-associated Fcgamma receptor IIa (FcgammaRIIa) and glycoprotein VI (GPVI)/FcR gamma-chain signaling pathways in platelets, its physiologic role in integrin alpha(IIb)beta3-mediated platelet function is unclear. In this study, we investigate the functional importance of PECAM-1 in murine platelets. Using PECAM-1-deficient mice, we show that the platelets have impaired "outside-in" integrin alpha(IIb)beta3 signaling with impaired platelet spreading on fibrinogen, failure to retract fibrin clots in vitro, and reduced tyrosine phosphorylation of focal adhesion kinase p125 (125FAK) following integrin alpha(IIb)beta3-mediated platelet aggregation. This functional integrin alpha(IIb)beta3 defect could not be attributed to altered expression of integrin alpha(IIb)beta3. PECAM-1-/- platelets displayed normal platelet alpha granule secretion, normal platelet aggregation to protease-activated receptor-4 (PAR-4), adenosine diphosphate (ADP), and calcium ionophore, and static platelet adhesion. In addition, PECAM-1-/- platelets displayed normal "inside-out" integrin alpha(IIb)beta3 signaling properties as demonstrated by normal agonist-induced binding of soluble fluoroscein isothiocyanate (FITC)-fibrinogen, JON/A antibody binding, and increases in cytosolic-free calcium and inositol (1,4,5)P3 triphosphate (IP3) levels. This study provides direct evidence that PECAM-1 is essential for normal integrin alpha(IIb)beta3-mediated platelet function and that disruption of PECAM-1 induced a moderate "outsidein" integrin alpha(IIb)beta3 signaling defect.     

The tetraspanin superfamily member CD151 regulates outside-in integrin alphaIIbbeta3 signaling and platelet function

The tetraspanin family member CD151 forms complexes with integrins and regulates cell adhesion and migration. While CD151 is highly expressed in megakaryocytes and to a lesser extent in platelets, its physiologic role in platelets is unclear. In this study, we investigate the physical and functional importance of CD151 in murine platelets. Immunoprecipitation/Western blot studies reveal a constitutive physical association of CD151 with integrin alpha(IIb)beta(3) complex under strong detergent conditions. Using CD151-deficient mice, we show that the platelets have impaired "outside-in" integrin alpha(IIb)beta(3) signaling with defective platelet aggregation responses to protease-activated receptor 4 (PAR-4) agonist peptide, collagen, and adenosine diphosphate (ADP); impaired platelet spreading on fibrinogen; and delayed kinetics of clot retraction in vitro. This functional integrin alpha(IIb)beta(3) defect could not be attributed to altered expression of integrin alpha(IIb)beta(3). CD151(-/-) platelets displayed normal platelet alpha granule secretion, dense granule secretion, and static platelet adhesion. In addition, CD151(-/-) platelets displayed normal "inside-out" integrin alpha(IIb)beta(3) signaling properties as demonstrated by normal agonist-induced binding of soluble fluorescein isothiocyanate (FITC)-fibrinogen, JON/A antibody binding, and increases in cytosolic-free calcium and inositol 1,4,5 triphosphate (IP(3)) levels. This study provides the first direct evidence that CD151 is essential for normal platelet function and that disruption of CD151 induced a moderate outside-in integrin alpha(IIb)beta(3) signaling defect.     

Activation of phospholipases A and C in human platelets exposed to epinephrine: role of glycoproteins IIb/IIIa and dual role of epinephrine.

Human platelets stimulated by epinephrine undergo enhanced turnover of phosphatidylinositol 4,5-bisphosphate, accumulate inositol trisphosphate, diacylglycerol, and phosphatidic acid, and phosphorylate a 47-kDa protein. All of these phenomena indicate stimulation of phospholipase C. These responses are blocked completely by inhibitors of alpha 2-adrenergic receptors (yohimbine), cyclooxygenase (aspirin or indomethacin), phospholipase A [2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid (ONO-RS-082)], Na+/H+ exchange [ethylisopropylamiloride (EIPA)], fibrinogen binding to glycoprotein IIb/IIIa (antibody A2A9), Ca2+/Mg+ binding (EDTA), or removal of fibrinogen. Epinephrine evokes (i) an increased turnover of ester-linked arachidonic acid in aspirin treated platelets that is inhibited by ONO-RS-082, EDTA, yohimbine, or the absence of fibrinogen and (ii) a rapid cytoplasmic alkalinization that is inhibited partially by blockage of cyclooxygenase activity and completely by A2A9 or EIPA. In contrast, when incubated with subaggregatory concentrations of the prostaglandin H2/thromboxane A2 analogue [(15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic acid (U46619) and epinephrine, aspirin-treated platelets show a potentiation of phospholipase C activation that is unaffected by the above inhibitors. We propose that epinephrine, in promoting exposure of glycoprotein IIb/IIIa sites for fibrinogen binding, leads to a cytoplasmic alkalinization, which, in conjunction with local shifts in Ca2+, promotes low-level activation of phospholipase A. The resulting free arachidonic acid is converted to cyclooxygenase products, which, potentiated by epinephrine, activate phospholipase C. This further amplifies the initial stimulatory response.     

Platelet HDL(3) binding sites are not related to integrin alpha(IIb)beta(3) (GPIIb-IIIa)

Early studies considered that fibrinogen receptor (glycoprotein [GP] IIb-IIIa or platelet integrin alpha(IIb)beta(3)) is the binding site for low-density lipoprotein (LDL) and high-density lipoprotein type 3 (HDL(3)). Recent data, however, do not support the hypothesis that the binding of LDL to human intact resting platelets is related to integrin alpha(IIb)beta(3). In this study we present evidence that platelet integrin alpha(IIb)beta(3) is also not involved in the interaction of HDL(3) and human intact resting platelets. Firstly, specific ligands for platelet integrin alpha(IIb)beta(3), such as fibrinogen, vitronectin, von Willebrand factor and fibronectin, were unable to inhibit the binding of HDL(3) to intact resting platelets. Secondly, the HDL(3) binding characteristics (K(d) and B(max) values), the activation of protein kinase C (PKC) and the inhibition of thrombin-induced inositoltriphosphate (IP(3)) formation and calcium (Ca(2+)) mobilization mediated by HDL(3) particles were similar in platelets from control subjects and patients with type I and type II Glanzmann's thrombasthenia, which are characterized by total and partial lack of GPIIb-IIIa and fibrinogen, respectively. In contrast, nitrosylation of tyrosine residues of HDL(3) by tetranitromethane fully abolished both the ability of particles to interact with its specific binding sites and the functional effects. Thirdly, polyclonal antibodies against the GPIIb-IIIa complex (edu-3 and 5B12), human antiserums against platelet alloantigens (anti-Bak(a/B) and anti-PL(A1/2)), anti-integrin subunits (anti-alpha(V) and anti-beta(3)), and a wide panel of monoclonal antibodies (mAbs) against well-known epitopes of GPIIb (M3, M4, M5, M6, M8 and M95-2b) and GPIIIa (P23-7, P33, P37, P40, and P97) did not affect the binding of HDL(3) particles to human intact resting platelets. Overall results show that neither the GPIIb-IIIa complex nor GPIIb or GPIIIa individually are the membrane binding proteins for HDL(3)on intact resting platelets.