Effect of prostacyclin analogue (carbacyclin) on the interaction of platelets with different collagen substrates. Inhibition of camp inccrease by collagens type I, III and IV

We investigated the effects of a stable prostacyclin analogue, carbacylcin, on the interaction of platelets with collagen substrates differing in thier ability to activate platelets: human collagens type I. III, IV and V (CI, CIII, CIV and CV), and commercial calf skin collagen type I (CSC). The total adhension was measured using ⁵¹Cr-labelled platelets, and quantitative morphometry of adherent platelets was performed by scanning electron microscopy (SEM). Carbacyclin in the concentrations inducing a 10-fold rise in platelets cAMP did not effect the adhension of platelets to weak substrates, CV and CSV, but reduced the adhesion to strong substrates, CIV and *by 49%) and CI/CIII (by 78%), which stimulated massive spreading and formation of surface-boud aggregates respectively. Carbacyclin inhibited all morphological manifestations of platelet activation associated with adhension: conversion of native discoid platelets to spherical ones on CSC; massive spreading on CIV; and aggregate formation on CI/CIII. Massive spreading and aggregation on a weak substrate (CSC) stimulated by arachidonic acid and thrombin was also inhibited by carbacyclin. Under the same concentration of angonists aggregation of platelets was more sensitive to the action of carbacyclin, than spreading. Strong collagen substrates CI, CIII and CIV, but not CV and gelatin, inhibited the carbacyclin-induced rise in platelet cAMP.     

Thrombocyte adhesion and aggregation on surfaces coated with human type-I, -III, -IV and -V collagens

Human collagens of type I, III, IV, and V (CI, CIII, CIV, and CV) can be localized in different anatomic structures of the vessel wall. To investigate the role of vascular collagenous components in mural thrombus formation, the authors studied platelet adhesion to the wells of Falcon culture plates coated with: a) monomeric CI, CIII, CIV, and CV; b) fibrillar CI and CIII, and c) amorphous CIV and CV. On monomeric and amorphous CV, only initial attachment takes place, i.e. platelets bind to the surface without subsequent spreading. Platelet adhesion on monomeric and amorphous CIV proceeds more actively: the total level of adhesion is substantially higher than on CV, with up to 75% of adherent platelets spread out and single unspread platelets from suspension attached to the upper surface of spread platelets. On monomeric and fibrillar CI/CIII, formation of large multi-layer (thrombi-like) aggregates, with spread platelets at the basis, takes place along with processes characteristic for adhesion on CIV/CV. On the contrary, only fibrillar but not monomeric CI and CIII induce platelet aggregation in suspension. The data suggest that the ability of CI and CIII to induce platelet aggregation is fully conditioned by the genetic type of collagen and requires a simultaneous multivalent platelet-collagen interaction, which can be achieved by surface immobilization of collagen or formation of fibrillar structures in suspension.     

A comparison of the inhibitory effects of prostacyclin and carbacyclin on platelet adhesion to collagen

The effect of carbacyclin, a chemically stable analogue of prostacyclin (PGI2), on the adhesion of platelets to collagen has been examined. The compound was compared to PGI2 which is unstable and rapidly hydrolysed to the inactive derivative, 6-oxo-PGF 1 alpha. The adhesion of 111Indium-labelled human platelets to collagen in the absence of platelet aggregation and secretion was measured. The cAMP level in the platelets was also monitored. Both PGI2 and carbacyclin inhibited platelet-collagen adhesion and caused a rise in the platelet cAMP level. Carbacyclin was approximately 15-fold less effective than PGI2, however, its effect was longer lasting, remaining constant for at least 30 minutes.     

A comparison of the inhibitory effects of prostacyclin and carbacyclin on platelet adhesion to collagen

The effect of carbacyclin, a chemically stable analogue of prostacyclin (PGI₂), on the adhesion of platelets to collagen has been examined. The compound was compared to PGI₂ which is unstable and rapidly hydrolysed to the inactive derivative, 6-oxo-PGF_1α. The adhesion of ¹¹¹Indium-labelled human plateles to collagen in the absence of platelet aggregation and secretion was measured. The cAMP level in the platelets was also monitored. Both PGI₂ and carbacyclin inhibited platelet-collagen adhesion and caused a rise in the platelet cAMP level. Carbacyclin was approximately 15-fold less effective than PGI₂, however, its effect was longer lasting, remaining constant for at least 30 minutes.     

Carbacyclin — A potent stable prost acyclin analogue for the inhibition of platelet aggregation

Carbacyclin is a chemically stable analogue of prostacyclin. As an inhibitor of platelet aggregation induced by ADP or collagen in vitro, carbacyclin is 0.03 times as active as prostacyclin in human, dog or rabbit plasma. Carbacyclin, like prostacyclin, reduces systemic arterial blood pressure (BP) in dogs, rabbits and rats and is not inactivated during passage through the pulmonary circulation. Further actions were investigated using a new ex vivo technique which allows rapid preparation of platelet-rich plasma and determination of platelet aggregation. In the dog, intravenous infusion of carbacyclin or prostacyclin inhibits platelet aggregation ex vivo with minimal effects on BP or heart rate. In the anaesthetised or conscious rabbit, carbacyclin and prostacyclin produces similar cardiovascular changes in doses producing an equivalent degree of platelet inhibition. In both rabbit and dog, carbacyclin is 0.1 times as active as prostacyclin in inhibiting ex vivo platelet aggregation. Platelet inhibition is maintained throughout the period of infusion of either compound (up to 3 h) yet is no longer apparent 10 min after terminating the infusion. Carbacyclin is thus a chemically-stable but metabolically-unstable analogue with a biological profile closely similar to prostacyclin.     

The role of thrombocyte prostanoids and products secreted by dense granules in the platelet attachment, spreading and aggregation on collagen substrates

The role of platelet prostanoids and substances released from dense bodies (ADP and serotonin) in the initial attachment, spreading and aggregation of platelets on surfaces coated with I, III, IV and V genetic types of collagen was investigated. A positive linear correlation was found to exist between thrombi-like aggregate formation on collagen substrates and platelet prostanoid synthesis. No correlation was established between platelet aggregate formation and 14C-serotonin release. The cyclooxygenase inhibitor indomethacin and the antagonists of PG endoperoxides and TXA2 (13-APA and BM 13.177) completely block thrombi-like aggregate formation. Neither 13-APA nor BM 13.177 affect platelet spreading, while indomethacin inhibits this process by 25%. The ADP-scavenger CP/CPK inhibits platelet aggregation and spreading by 25-30%. The inhibitors of cyclooxygenase and CP/CPK do not influence the initial attachment of platelets. The data obtained suggest that thrombi-like aggregate formation on collagen substrates is mediated by the synthesis of PG endoperoxides and TXA2; however, in platelet spreading this synthesis plays a limited role. Spreading and aggregation of platelets on collagen substrates is only partly mediated by ADP and serotonin. Initial attachment of platelets does not depend on ADP and serotonin release and PG endoperoxide/TXA2 synthesis.     

Modulation of platelet activation by native DNA.

Native DNA (dsDNA) was found to induce the aggregation of isolated human platelets and the release of platelet 5HT; this activation was inhibited by both theophylline and TYA, suggesting a role for cAMP and metabolic products formed from arachidonate. By contrast, nonaggregating amounts of dsDNA inhibited platelet activation induced by collagen or thrombin. This inhibition, which could be overcome by use of greater amounts of the stimulatory agents, was not associated with the loss of platelet viability. Activation of platelets by dsDNA was not observed in plasma or in isolated platelet systems to which small amounts of cell-free plasma were added. However, dsDNA maintained in plasma its ability to inhibit platelet aggregation induced by collagen and thrombin. RNA and single-stranded DNA failed to induce platelet aggregation or release of 5HT and to block the platelet activation stimulated by dsDNA. Further, dsDNA did not significantly inhibit platelet aggregation in platelet-rich plasma stimulated by ADP or epinephrine. These data implicate dsDNA as a selective and potentially important activator and modulator of platelet responsiveness.     

Inhibitory effect of ginkgolide B on platelet aggregation in a cAMP- and cGMP-dependent manner by activated MMP-9

Extracts from the leaves of the Ginkgo biloba are becoming increasingly popular as a treatment that is claimed to reduce atherosclerosis, coronary artery disease, and thrombosis. In this study, the effect of ginkgolide B (GB) from Ginkgo biloba leaves in collagen (10 microg/ml)- stimulated platelet aggregation was investigated. It has been known that human platelets release matrix metalloproteinase- 9 (MMP-9), and that it significantly inhibited platelet aggregation stimulated by collagen. Zymographic analysis confirmed that pro-MMP-9 (92-kDa) was activated by GB to form an MMP-9 (86-kDa) on gelatinolytic activities. And then, activated MMP-9 by GB dose-dependently inhibited platelet aggregation, intracellular Ca2+ mobilization, and thromboxane A2 (TXA2) formation in collagen-stimulated platelets. Activated MMP-9 by GB directly affects down-regulations of cyclooxygenase-1 (COX-1) or TXA2 synthase in a cell free system. In addition, activated MMP-9 significantly increased the formation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which have the anti-platelet function in resting and collagen-stimulated platelets. Therefore, we suggest that activated MMP-9 by GB may increase the intracellular cAMP and cGMP production, inhibit the intracellular Ca2+ mobilization and TXA2 production, thereby leading to inhibition of platelet aggregation. These results strongly indicate that activated MMP-9 is a potent inhibitor of collagen-stimulated platelet aggregation. It may act a crucial role as a negative regulator during platelet activation.     

Feverfew--an antithrombotic drug?

The effects of an extract of the plant feverfew on the interaction of platelets with surfaces coated with human collagens of type III and IV (CIII, CIV), and on the integrity of the endothelial cell (EC) monolayer in perfused rabbit aorta were studied. It was shown that feverfew extract (FE) inhibited the deposition of [51Cr]-labelled platelets on both CIII and CIV in a dose-dependent way. Similar concentrations of FE were needed to inhibit formation of surface-bound aggregates in CIII and platelet spreading on CIV in both platelet-rich plasma and GFP. When aorta segments were perfused in situ with a physiological salt solution, the addition of FE to the solution protected the EC monolayer from spontaneous injury. The results indicate that feverfew may have antithrombotic potential in addition to its claimed benefit in fever, migraine and arthritis.     

Mechanisms of inhibition of vascular-platelet hemostasis by salivary gland secretion of the medicinal leech Hirudo medicinalis

The mechanism of inhibition of the vascular-platelet stage of hemostasis by medicinal leech salivary gland secretion was studied. It was shown that the secretion blocks platelet adhesion on the surface of collagens belonging to different genetic classes, inhibits the primary attachment of platelets and completely suppresses their spreading on collagen surface. Whatever its antithrombin activity, the leech secretion inhibits platelet aggregation stimulated by various inductors, e. g., ADP, prostaglandin endoperoxide analog U-46619, Ca2+ ionophore A23187, arachidonic acid. The secretion possessing the antithrombin activity causes a greater inhibition of the thrombin-stimulated aggregation than that devoid of this activity. Leech secretion stimulates adenylate cyclase of platelet membranes in a receptor-mediated fashion and increases the level of cAMP. The active substance is a low molecular weight, thermostable trypsin-resistant fraction of the secretion. Stimulation of adenylate cyclase is not mediated by adenosine receptors. It is supposed that the mechanism of this activating effect involves platelet prostaglandin receptors.     

Stimulation of spreading and aggregation of platelets on immobilized type V collagen: mechanisms depending on Ca2+ and protein kinase C

The effects of phorbol ester (PMA) and stable prostaglandin endoperoxide analog (U46619) on platelet interaction with a surface coated with monomeric type V collagen (CV substrate) and free Ca2+ concentration in platelet cytoplasm ([Ca2+]in) have been studied. In the absence of PMA and U46619, the discoid and spherical platelets from suspension are attached to CV substrate but are incapable of spreading and aggregation on the substrate. An addition of PMA (0.15-1.5 nM) or U46619 (1.5 microM) to the reaction mixture stimulates platelet spreading and the formation of multilayer (thrombi-like) aggregates on CV substrate. Using the fluorescent probe Quin 2, it was found that U46619 (0.1 microM) increases [Ca2+]in from the basal level (100-120 mM) to 600 nM. PMA (0.75-15 nM) exerts only a slight effect increasing [Ca2+]in by 30-40 nM. The data obtained suggest that the PMA-induced spreading and aggregation of platelets on CV substrate can occur via activation of protein kinase C at relatively low [Ca2+]in values. These results also testify to the existence of a substrate-independent mechanism of spreading of platelets activated in suspensions by soluble inducers.     

Interrelation of platelet aggregation, release reaction and thromboxane A2 production

Aggregation of platelets, stimulated by different agonists, was inhibited by omitting sample stirring or by preincubation of platelets with a monoclonal antibody against glycoproteins IIb-IIIa or with a pentapeptide containing the sequence Arg-Gly-Asp-Ser. In platelets stimulated by collagen, ADP and epinephrine, the inhibition of aggregation paralleled a reduction of both release reaction and thromboxane A2 formation. When thrombin was the stimulus, ATP release and thromboxane A2 production were unaffected (or only slightly modified) by the inhibition of platelet aggregation. These data add further evidence to the hypothesis that aggregation supports the activation of platelets stimulated by weak agonists.     

Platelet-collagen interaction. Inhibition by a monoclonal antibody raised against collagen receptor

Monoclonal antibodies to the purified platelet type I collagen receptor were produced to study platelet receptor function. The antibody specifically reacted with the platelet receptor in immunoblot experiments. The IgG purified from the monoclonal antibodies and isolated Fab' fragments inhibited the binding of radiolabeled alpha 1(I) chain to washed platelets competitively. Soluble and fibrillar type I collagen-induced platelet aggregations were inhibited by purified IgG suggesting that soluble and fibrillar collagens shared a common receptor. The adhesion of platelets to an artificial collagen matrix was also inhibited by the monoclonal antibody. However, adenosine diphosphate-induced platelet aggregation was not inhibited by the same amount of IgG that inhibited collagen-induced platelet aggregation. The results suggest that collagen-induced platelet aggregation is mediated through the interaction of collagen with the platelet receptor.     

An antiplatelet peptide, gabonin, from Bitis gabonica snake venom.

Interaction of fibrinogen with its receptors (glycoprotein IIb/IIIa complex) on platelet membranes leads to platelet aggregation. By means of gel filtration, CM-Sephadex C-50, and reverse-phase HPLC, an antiplatelet peptide, gabonin, was purified from the venom of Bitis gabonica. The purified protein migrates as a 21,100-Da polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions and as a 11,000-Da peptide in the presence of beta-mercaptoethanol, indicating that gabonin is a disulfide-linked dimer. It is a polypeptide consisting of about 84 amino acid residues, rich in Asp, Pro, and half-cystine. Gabonin dose-dependently inhibited human platelet aggregation stimulated by ADP, collagen, U46619, or thrombin in preparations of platelet-rich plasma and platelet suspension (IC50 = 340-1600 nM). It also blocked platelet aggregation of whole blood. However, it apparently did not affect the initial shape change and only slightly reduced ATP release caused by aggregation agonists. Gabonin did not inhibit the rise of cytosolic calcium in Quin-2-loaded platelets stimulated by thrombin. In addition, gabonin dose-dependently inhibited fibrinogen-induced aggregation of elastase-treated platelets. In conclusion, gabonin inhibits platelet aggregation mainly through the blockade of fibrinogen binding toward fibrinogen receptors of the activated platelets.     

Mitochondrial hyperpolarization during chronic complex I inhibition is sustained by low activity of complex II,III, IV and V

The mitochondrial oxidative phosphorylation (OXPHOS) system consists of four electron transport chain (ETC) complexes (CI–CIV) and the F_oF₁-ATP synthase (CV), which sustain ATP generation via chemiosmotic coupling. The latter requires an inward-directed proton-motive force (PMF) across the mitochondrial inner membrane (MIM) consisting of a proton (ΔpH) and electrical charge (Δψ) gradient. CI actively participates in sustaining these gradients via trans-MIM proton pumping. Enigmatically, at the cellular level genetic or inhibitor-induced CI dysfunction has been associated with Δψ depolarization or hyperpolarization. The cellular mechanism of the latter is still incompletely understood. Here we demonstrate that chronic (24 h) CI inhibition in HEK293 cells induces a proton-based Δψ hyperpolarization in HEK293 cells without triggering reverse-mode action of CV or the adenine nucleotide translocase (ANT). Hyperpolarization was associated with low levels of CII-driven O₂ consumption and prevented by co-inhibition of CII, CIII or CIV activity. In contrast, chronic CIII inhibition triggered CV reverse-mode action and induced Δψ depolarization. CI- and CIII-inhibition similarly reduced free matrix ATP levels and increased the cell's dependence on extracellular glucose to maintain cytosolic free ATP. Our findings support a model in which Δψ hyperpolarization in CI-inhibited cells results from low activity of CII, CIII and CIV, combined with reduced forward action of CV and ANT.     

Platelet aggregation may not be a prerequisite for collagen-stimulated platelet generation of nitric oxide

By determining the sum of the supernatant concentrations of nitrite and nitrate the stimulated generation of nitric oxide (NO) by human washed platelets induced by a range of fibrillar collagen concentrations (0.0156-25 microg ml(-1)) was investigated. Platelet serotonin (5-hydroxytryptamine, 5-HT) efflux and platelet aggregation were also measured. Under resting conditions (0 microg ml(-1) collagen) platelet NO release was equivalent to 1.06+/-0.17 nmol per 10(8) platelets. Maximal NO release, equivalent to 2.1+/-0. 37 nmol per 10(8) platelets, was observed with only 0.0625 microg ml(-1) collagen (P<0.02, stimulated vs. resting release), higher collagen concentrations producing no further increases in platelet NO output. By contrast, maximal platelet aggregation and 5-HT efflux did not occur until collagen concentrations of 2.5 microg ml(-1) and 10-25 microg ml-1), respectively, had been achieved. L-NAME (1 mmol l(-1)) and L-NMMA (1 mmol l(-1)) inhibited stimulated platelet NO generation by 78+/-6% and 72%, respectively. Contrasting with fibrillar collagen, fibrillar beta-amyloid protein had no effect on platelet NO generation, or on 5-HT efflux or aggregation. These data perhaps indicate that NO generation by human platelets is stimulated by concentrations of fibrillar collagen insufficient to elicit an aggregatory response. Such a mechanism could operate in vivo to inhibit platelet aggregation which might otherwise be induced by low concentrations of circulating agonists.     

Adherence of platelets under flow conditions results in specific phosphorylation of proteins at tyrosine residues

Collagen is a powerful platelet activating agent that promotes adhesion and aggregation of platelets. To differentiate the signals generated in these processes we have analyzed the tyrosine phosphorylation occurring in platelets after activation with collagen in suspension or under flow conditions. For the suspension studies, washed platelets were activated with different concentrations of purified type I collagen (ColI). Studies under flow conditions were performed using two different adhesive substrata: ColI and endothelial cells extracellular matrix (ECM). Coverslips coated with ColI or ECM were perfused through a parallel-plate perfusion chamber at 800 s(-1) for 5 min. After activation of platelets either in suspension or by adhesion, samples were solubilized and proteins were resolved by electrophoresis. Tyrosine-phosphorylated proteins were detected in immunoblots by specific antibodies. Activation of platelet suspensions with collagen induced tyrosine phosphorylation before aggregation could be detected. Profiles showing tyrosine-phosphorylated proteins from platelets adhered on ColI or on ECM were almost identical and lacked proteins p95, p80, p66, and p64, which were present in profiles from platelets activated in suspension. The intensity of phosphorylation was quantitatively weaker in those profiles from platelets adhered on ECM. Results from the present work indicate that activation of platelets in suspension or by adhesion induces differential tyrosine phosphorylation patterns. Phosphorylation of proteins p90 and p76 may be related to early activation events occurring during initial contact and spreading of platelets. Considering that adhesion is the first step of platelet activation, studies on signal transduction mechanisms under flow conditions may provide new insights to understand the signaling processes taking place at earliest stages of platelet activation.     

Adherence of Platelets Under Flow Conditions Results in Specific Phosphorylation of Proteins at Tyrosine Residues

Collagen is a powerful platelet activating agent that promotes adhesion and aggregation of platelets. To differentiate the signals generated in these processes we have analyzed the tyrosine phosphorylation occurring in platelets after activation with collagen in suspension or under flow conditions. For the suspension studies, washed platelets were activated with different concentrations of purified type I collagen (Coll). Studies under flow conditions were performed using two different adhesive substrata: Coll and endothelial cells extracellular matrix (ECM). Coverslips coated with Coll or ECM were perfused through a parallel-plate perfusion chamber at 800s^?1 for 5 min. After activation of platelets either in suspension or by adhesion, samples were solubilized and proteins were resolved by electrophoresis. Tyrosine-phosphorylated proteins were detected in immunoblots by specific antibodies. Activation of platelet suspensions with collagen induced tyrosine phosphorylation before aggregation could be detected. Profiles showing tyrosine-phosphorylated proteins from platelets adhered on Coll or on ECM were almost identical and lacked proteins p95, p80, p66, and p64. which were present in profiles from platelets activated in suspension. The intensity of phosphorylation was quantitatively weaker in those profiles from platelets adhered on ECM. Results from the present work indicate that activation of platelets in suspension or by adhesion induces differential tyrosine phosphorylation patterns. Phosphorylation of proteins p90 and p76 may be related to early activation events occurring during initial contact and spreading of platelets. Considering that adhesion is the first step of platelet activation, studies on signal transduction mechanisms under flow conditions may provide new insights to understand the signaling processes taking place at earliest stages of platelet activation.     

Antioxidants change platelet responses to various stimulating events

The role of platelets in hemostasis may be influenced by alteration of the platelet redox state—the presence of antioxidants and the formation of reactive oxygen and nitrogen species. We investigated the effects of two antioxidants, resveratrol and trolox, on platelet activation. Trolox and resveratrol inhibited aggregation of washed platelets and platelet-rich plasma activated by ADP, collagen, and thrombin receptor-activating peptide. Resveratrol was a more effective agent in reducing platelet static and dynamic adhesion in comparison with trolox. The antioxidant capacity of resveratrol was, however, the same as that of trolox. After incubation of platelets with antioxidants, the resveratrol intraplatelet concentration was about five times lower than the intracellular concentration of trolox. Although both antioxidants comparably lowered hydroxyl radical and malondialdehyde production in platelets stimulated with collagen, TxB₂ levels were decreased by resveratrol much more effectively than by trolox. Cyclooxygenase 1 was inhibited by resveratrol and not by trolox. Our data indicate that antioxidants, apart from nonspecific redox or radical-quenching mechanisms, inhibit platelet activation also by specific interaction with target proteins. The results also show the importance of studying platelet activation under conditions of real blood flow in contact with reactive surfaces, e.g., using dynamic adhesion experiments.     

Adhesion of human platelets to immobilized trimeric collagen

Human platelets adhere to trimeric Type 1 chick collagen that was covalently linked to plastic slides, providing the basis for a well- defined quantitative assay. The number of platelets that adhere is a function both of platelet concentration and of collagen density on the slides. In contrast with other in vitro assays using collagen that is not covalently linked to the substratum, we found no platelet-platelet aggregation. Adhesion was absolutely dependent on Mg2+, whereas Ca2+ was ineffective. Native trimeric collagen conformation was required for adhesion, since platelets did not bind to slides containing heat- denatured collagen, or isolated alpha 1(1) or or alpha 2(1) chains. Modifications of collagen oligosaccharides had no effect on adhesion. Adhesion was inhibited by cytochalasin D but was not affected by prostaglandin E1, apyrase, acetylsalicylic acid, or theophylline. Because this assay measures platelet-collagen adhesion in the absence of platelet-platelet aggregation, it should facilitate identification of the platelet surface components that directly mediate this adhesion.