Compound 48/80 is a potent inhibitor of phospholipase C and a dual modulator of phospholipase A2 from human platelet

Compound 48/80 inhibited phosphatidylinositol-specific phospholipase C activity from human platelets. Whereas 1 microgram/ml of compound 48/80 slightly stimulated Ca2+-dependent phospholipase A2, higher concentrations led to dose-dependent inhibition of this platelet enzyme. This biphasic effect was confirmed with phospholipases A2 purified from rat liver and human synovial fluid. The aggregation of human platelets induced by ADP and PAF-acether was inhibited by compound 48/80, whereas the aggregation induced by ionophore A23187 was not modified by this compound. These results demonstrate that the inhibition of platelet aggregation by compound 48/80 is not due solely to effects on calmodulin as previously reported, but that inhibition of phospholipases and probably arachidonate mobilization may also be involved.     

Effects of C-reactive protein on platelet function. III. The role of cAMP, contractile elements, and prostaglandin metabolism in CRP-induced inhibition of platelet aggregation and secretion.

It was previously demonstrated that C-reactive protein (CRP) inhibits platelet aggregation and release reactions, activation of platelet factor 3, and platelet-dependent clot retraction. Multiple considerations including selective inhibition of secondary wave aggregation suggested that CRP exerted its inhibitory effects by interfering with the release of endogenous ADP. In the present investigation, CRP was found by direct assay to inhibit the release of endogenous ADP and/or serotonin concomitant with inhibition of platelet aggregation stimulated by ADP, epinephrine, thrombin, and AHGG. CRP did not induce an increase in the basal level of platelet cAMP, suggesting independence of a direct effect upon this mediator system. Furthermore, CRP did not inhibit the aggregation and secretion induced by the antibiotic ionophore A23187, suggesting the absence of a direct effect upon the activation of platelet contractile elements. By contrast, CRP did inhibit both thrombin-induced release of malondialdehyde, a prostaglandin endoperoxide nonprostanoate endproduct, and platelet aggregation induced by the prostaglandin endoperoxide precursor arachidonic acid. These data, therefore, raise the possibility that CRP inhibits platelet reactivities by interfering with an aspect of porstaglandin metabolism, and that this occurs subsequent to the hydrolytic accumulation of arachidonic acid and prior to the movement of calcium from the platelet dense tubules. These studies support the concept that CRP serves to modulate platelet reactivities during acute inflammatory reactions.     

Platelet aggregation induced by platelet-activating factor is suppressed by cystine protease inhibitor

The effect of NCO-700, a cystine protease inhibitor, on platelet-activating factor-induced platelet aggregation was determined. A newly synthesized cystine protease inhibitor (calcium-activated neutral protease and cathepsin B inhibitor), NCO-700 (bis[ethyl (2R, 3R)-3-[(S)-methyl-1-[4-(2,3,4- trimethoxyphenylmethyl) piperazine-1-ylcarbonyl]butylcarbonyl]oxiran-2-carboxy late]sulfate), inhibited platelet-activating factor-induced platelet aggregation. The inhibition was dependent on the preincubation time with NCO-700 and on the concentration of the inhibitor. The release of serotonin was also inhibited almost completely by the 20-min preincubation with 10(-4) M NCO-700. Leupeptin also inhibited platelet-activating factor-induced platelet aggregation. But calcium-activated neutral protease inhibitor did not inhibit it. These observations suggest that NCO-700-sensitive protease(s) such as cystine protease may contribute to platelet aggregation induced by platelet-activating factor.     

Cleaved forms of C-reactive protein are associated with platelet inhibition

C-reactive protein (CRP) is the prototypic acute phase reactant and serves clinically as a marker of inflammation and tissue destruction. When native CRP pentamer was incubated with Streptomyces griseus protease, a newly formed and transient ability to inhibit platelet aggregation stimulated by adenosine diphosphate or collagen was often elicited early during the course of enzymatic digestion. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses of the digests revealed that platelet inhibitory activity correlated with altered electrophoretic mobility and reductions in subunit and pentameric m.w. Minimally degraded forms of CRP were also isolated "de novo" from inflammatory fluids and, like their enzyme degraded counterparts, inhibited platelet activation. Dissociation of degraded CRP with SDS followed by the removal of SDS resulted in the separation fragments which inhibited platelet function. We propose that in a degradative environment, such as at sites of inflammation/tissue damage or through the action of serum proteases, CRP may transitorily down-regulate the platelet.     

Ca2+ signaling in the transformation of promastigotes to axenic amastigotes of Leishmania donovani

Three acidic phospholipases A₂ from Indian cobra (Naja naja naja) venom inhibited platelet aggregation in platelet rich plasma induced separately by ADP, collagen and epinephrine with different potencies. The order of inhibition was epinephrine > collagen > ADP. They did not inhibit platelet aggregation induced by arachidonic acid (10 M). The inhibition was dependent on concentration of the protein and the time of incubation of the phospholipases A₂ with platelet rich plasma. Parabromophenacyl bromide modified PLA₂ enzymes lost their enzymatic activity as well as platelet aggregation inhibition activity suggesting the involvement of catalytic function in platelet aggregation inhibitory activity.     

Effects of C-reactive protein on human neutrophil granulocytes challenged with N-formyl-methionyl-leucyl-phenylalanine and platelet-activating factor

The effects of C-reactive protein (CRP), the prototypical acute-phase reactant were studied on human polymorphonuclear leukocytes (PMNL) challenged with N-formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF). CRP at 8-64 micrograms/ml concentrations inhibited degranulation and superoxide production by PMNL in time-, and dose-dependent manner and stabilized PMNL membranes against the lytic effect of lysophosphatidylcholine. CRP was also capable of binding PAF and in lesser extent fMLP. Furthermore, CRP, 32 micrograms/ml, diminished specific binding of [3H]-fMLP and [3H]-PAF to PMNL. These findings imply that CRP may play an important protective role during the early phase of acute inflammatory reactions.     

Modulation of stimulus-dependent human platelet activation by C-reactive protein modified with active oxygen species

C-reactive protein (CRP) is one of the most characteristic acute phase proteins which appear in the serum during certain inflammatory diseases. We report here that human CRP acquired the ability to augment platelet reactivity when treated with an Fe2+ (Cu2+)-ascorbate system. CRP modified by such treatment showed no appreciable activation of platelets in the absence of platelet activators such as platelet-activating factor, thrombin, or ADP. However, in the presence of the modified-CRP, irreversible activation of platelets occurred with sub-optimal doses of platelet-activating factor and other stimulatory agents for platelets. CRP without any treatment did not show any modulating activity. Each component of the Fe2+-ascorbate system was required for modification of CRP, suggesting that CRP was modified through an oxidative process. The modification of the CRP structure was confirmed by the change in the fluorescence spectrum of 8-anilino-1-naphthalene sulfonate complexed with CRP, the increased susceptibility of CRP to proteolytic enzymes and the altered reactivity to anti-CRP mAb. We also found an inactivating system for the modified CRP in plasma. The modified human CRP did not show any modulating activity toward rabbit platelets, suggesting that the activity is species specific.     

Bioactive amide of prostaglandin E1 and ethanolamine plasmalogen analog of platelet-activating factor inhibits several pathways of human platelet aggregation

The influence of an amide of prostaglandin E1 and ethanolamine plasmalogen platelet-activating factor analog 1-O-alk-1;-enyl-2-acetyl-sn-glycero-3-phospho-(N-11alpha, 15alpha-dioxy-9-keto-13-prostenoyl)ethanolamine (PGE1-PPAF) on platelet-activating factor (PAF)-, ADP-, and thrombin-induced human platelet aggregation has been studied. It was found that PGE1-PPAF inhibits the PAF-, ADP-, and thrombin-induced platelet aggregation in platelet-rich plasma. 1-O-alk-1;-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine inhibited PAF-induced aggregation up to 50% but had no influence on platelet aggregation induced by ADP or thrombin. The ethanolamine plasmalogen analog of PAF 1-O-alk-1;-enyl-2-acetyl-sn-glycero-3-phospho-(N-palmitoyl)ethanolami ne, having a palmitoyl residue instead of PGE1, did not inhibit platelet aggregation induced by PAF, ADP, or thrombin. We propose that inhibition of human platelet aggregation by PGE1-PPAF is mediated by its action on platelet PAF-receptors and the adenylate cyclase system.     

Human endothelial cells inhibit granulocyte aggregation in vitro

Granulocyte aggregation in response to circulating or locally released inflammatory mediators may cause vascular injury. The factors that regulate the granulocyte aggregation response and prevent its occurrence are not defined. We found that primary monolayers of human endothelial cells (EC) derived from umbilical veins released products that inhibited granulocyte aggregation. When polymorphonuclear leukocytes (PMN) and EC were incubated together, the subsequent aggregation response to N-formyl-methionyl-leucyl-phenylalanine (fMLP) was inhibited by 40 to 60%, depending in part on the duration of incubation and the concentration of the agonist. Suspension of the granulocytes in albumin-containing buffer that had been rocked with EC monolayers had a similar effect, demonstrating that the EC release a soluble product that modulates the aggregation response. The fMLP concentration-response curve was shifted downward and to the right by EC. Incubation of the granulocytes with endothelial monolayers for various times indicated that the inhibition was maximal at 2 to 3 min, and the PMN responsiveness returned to control over the next 15 min. The inhibiting effect was not selectively directed against fMLP, because incubation of PMN with EC or suspending the PMN in supernatants from endothelial monolayers also inhibited aggregation stimulated by platelet-activating factor, leukotriene B4, and C5a desarg. Release of the inhibitory activity by EC was attenuated by indomethacin, suggesting that the activity is in part due to a cyclooxygenase pathway product. Prostacyclin (PGI2), an eicosanoid produced by EC via the cyclooxygenase pathway, inhibited granulocyte aggregation; however, PGI2 was much less potent as an inhibitor of PMN aggregation than of platelet aggregation. Furthermore, the concentration of PGI2 in buffer that had been incubated with EC was not sufficient to account for the magnitude of the PMN inhibition. The concentration of prostaglandin E2 (PGE2) was also insufficient to completely account for the inhibition. EC that had been treated with indomethacin or aspirin, which blocked the release of PGI2 and PGE2, retained the partial ability to release an activity that blunted granulocyte aggregation; this inhibiting activity was stable at 37 degrees C for 60 min. The results indicate that human EC have the biologic potential to modulate granulocyte aggregation stimulated by inflammatory mediators, and the activity is only partly due to PGI2 and other cyclooxygenase products of arachidonic acid.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of C-reactive protein on platelet function. I. Inhibition of platelet aggregation and release reactions.

C-reactive protein (CRP) is an acute phase reactant which shares numerous functional characteristics with the immunoglobulins. In the present study CRP was found to inhibit the aggregation of human platelets stimulated by either modified human immunoglobulin or thrombin. This effect did not involve chelation of calcium or cytotoxicity, and was overcome by larger amounts of the aggregating agents. CRP also inhibited the activation but not the activity of platelet factor 3 and the release of beta-glucuronidase. Thus, CRP can inhibit multiple platelet reactivities. We suggest that this property of CRP may play an important role in the control of platelet responsiveness during reactions of inflammation, defense, and repair.     

Inhibition of epinephrine-induced platelet aggregation by a derivative of wheat germ agglutinin

A nonagglutinating derivative of wheat germ agglutinin has been prepared and used as a probe to explore the initial events in platelet activation. The lectin derivative had no effect on platelet aggregation by adenosine diphosphate, collagen, ristocetin, wheat germ agglutinin or trypsin but aggregation induced by epinephrine or thrombin was inhibited. Unlike thrombin, the inhibition of aggregation by the derivative could not be overcome by increasing the concentration of epinephrine. The derivative did not affect the binding of [³H]dihydroergocryptine to platelets. A 74,000 dalton protein isolated from platelet membranes by lectin affinity chromatography strongly inhibited platelet activation by thrombin but not by epinephrine. The receptors for thrombin and for epinephrine on platelets are different but they are closely linked.     

Platelet aggregation and thromboxane release induced by arachidonic acid, collagen, ADP and platelet-activating factor following low dose acetylsalicylic acid in man

The present study was undertaken in order to characterize the dose-dependent nature of acetylsalicylic acid (ASA) on platelet aggregation and plasma thromboxane B2 (TXB2) release in healthy volunteers. Volunteers received either 25, 50, 100 or 500 mg daily for five consecutive days. At the end of the five day period, all dosages of ASA were capable of completely suppressing TXB2 production and arachidonic acid-induced platelet aggregation. At that time, the second phase of ADP-induced aggregation was also blocked. However, while the inhibition following 500 mg ASA was complete after 24 hours, total inhibition with 100, 50 and 25 mg was attained only after two, three and four days, respectively, indicating the cumulative effect of ASA on platelets. Aggregation induced by collagen was also inhibited dose-dependently- yet slower and at no time complete. ASA had no inhibitory effect on aggregation by platelet-activating factor (PAF). It is concluded that a daily dose of 50 mg ASA would suffice in blocking platelet TXA2 production and aggregation induced by most physiological agents.     

Rabbit platelet calcium ATPase differs from the human erythrocyte (Ca2+ + Mg2+)-ATPase in its response to three purified phospholipases A2, exogenous phospholipids and calmodulin

Human erythrocyte (Ca2+ + Mg2+)-ATPase and calcium ATPase of rabbit platelets were compared by their responses to a variety of treatments. These included three purified phospholipases A2 (acidic, neutral and basic) from Agkistrodon halys blomhoffii, as well as several phospholipids and lysophospholipids. The erythrocyte enzyme was stimulated 2-3-fold by all three phospholipases with maximal stimulation occurring at different concentrations of the three enzymes. The basic phospholipase was the most potent, followed by the neutral and acidic enzymes in that order. The calcium ATPase activity of the platelet was also stimulated by phospholipase treatment, but only by 10-20%. The stimulatory activity was attributable to hydrolysis of a very small portion of the total membrane phospholipid. Inactivation of the phospholipases by heating or chemical modification with p-bromophenacyl bromide abolished their ability to stimulate. Addition of polyphosphoinositides stimulated both ATPases. However, another acidic phospholipid, lysophosphatidic acid, stimulated only the erythrocyte enzyme and failed to affect the platelet calcium ATPase. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) had no effect on either enzyme, while the platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), its lyso compound and lysoPC inhibited both ATPases. Calmodulin stimulated the erythrocyte enzyme, but did not affect the platelet calcium ATPase. These results demonstrate that the protein-lipid interactions operative in the erythrocyte and platelet calcium ATPases are quite different.     

Antiplatelet actions of aporphinoids from Formosan plants

Seventeen aporphines were tested for antiplatelet activity. L-(+)-hemovine HCl and 7-hydroxydehydrothalicsimidine strongly inhibited platelet aggregation induced by adenosine 5'-diphosphate (ADP), arachidonic acid (AA), collagen, and platelet-activating factor (PAF). The latter showed the strongest antiplatelet activity with an IC50 of 70.4 microM against AA-induced platelet aggregation.     

Platelet aggregation and thromboxane release induced by arachidonic acid, collagen, ADP and platelet-activating factor following low dose acetylsalicylic acid in man

The present study was undertaken in order to characterize the dose-dependent nature of acetylsalicylic acid (ASA) on platelet aggregation and plasma thromboxane B₂ (TXB₂) release in healthy volunteers. Volunteers received either 25, 50, 100 or 500 mg daily for five consecutive days. At the end of the five day period, all dosages of ASA were capable of completely suppressing TXB₂ production and arachidonic acid-induced platelet aggregation. At that time, the second phase of ADP-induced aggregation was also blocked. However, while the inhibition following 500 mg ASA was complete after 24 hours, total inhibition with 100, 50 and 25 mg was attained only after two, three and four days, respectively, indicating the cumulative effect of ASA on platelets. Aggregation induced by collagen was also inhibited dose-dependently- yet slower and at no time complete. ASA had no inhibitory effect on aggregation by platelet-activating factor (PAF). It is concluded that a daily dose of 50 mg ASA would suffice in blocking platelet TXA₂ production and aggregation induced by most physiological agents.     

Action mechanism of the platelet aggregation inducer and inhibitor from Echis carinatus snake venom

Platelet aggregation inducer and inhibitor were isolated from Echis carinatus snake venom. The venom inducer caused aggregation of washed rabbit platelets which could be inhibited completely by heparin or hirudin. The venom inducer also inhibit both the reversibility of platelet aggregation induced by ADP and the disaggregating effect of prostaglandin E₁ on the aggregation induced by collagen in the presence of heparin. The venom inhibitor decreased the platelet aggregation induced by collagen, thrombin, ionophore A23187, arachidonate, ADP and platelet-activating factor (PAF) with an IC₅₀ of around 10 μg/ml. It did not inhibit the agglutination of formaldehyde-treated platelets induced by polylysine. In the presence of indomethacin or in ADP-refractory platelets or thrombin-degranulated platelets, the venom inhibitor further inhibited the collagen-induced aggregation. Fibrinogen antagonized competitively the inhibitory action of the venom inhibitor in collagen-induced aggregation. In chymotrypsin-treated platelets, the venom inhibitor abolished the aggregation induced by fibrinogen. It was concluded that the venom inducer caused platelet aggregation indirectly by the conversion of prothrombin to thrombin, while the venom inhibitor inhibited platelet aggregation by interfering with the interaction between fibrinogen and platelets.     

Cooperativity between platelet-activating factor and collagen in platelet aggregation

Cell lysate obtained from cultured vascular endothelial cells contained a substance which induced platelet aggregation. This substance was identified as a phospholipid, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor; PAF), by thin-layer chromatography, phospholipase A2 digestion, inhibition by a specific antagonist, CV-3988, and agonist-specific refractory state. It was further found that PAF and collagen together induced extensive aggregation of platelets even with the concentrations by which each agonist alone could not induce aggregation of platelets at all.     

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

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

Core aldehydes of alkyl glycerophosphocholines in atheroma induce platelet aggregation and inhibit endothelium-dependent arterial relaxation.

Plaque disruption with superimposed thrombosis is considered to be responsible for precipitating acute coronary syndrome. We identified sn-1-alkyl- and sn-1-acyl-type glycerophosphocholine (GroPCho) core aldehydes from human atheromas and demonstrated their activities on platelets and arteries. The naturally occurring core aldehydes were identified and quantified in relation to synthetic standards by high performance liquid chromatography with on-line electrospray mass spectrometry. 1-O-Hexadecyl-2-(5-oxovaleroyl)-sn-GroPCho (C(5) alkyl GroPCho core aldehyde), occurring in atheroma at less than 0.1% of total phosphatide, induced aggregation of washed rabbit platelets (50% effective dose was approximately 50 nM). Aggregations induced by C(5) alkyl GroPCho core aldehydes were completely inhibited by two different platelet-activating factor receptor antagonists. 1-Palmitoyl-2-(5-oxovaleroyl)-sn-GroPCho (C(5) acyl GroPCho core aldehyde) induced platelet shape change, but not aggregation. By contrast, 10 microM C(5) alkyl and C(5) acyl GroPCho core aldehydes both inhibited endothelium-dependent relaxation of rabbit artery by 50% (endothelium-independent relaxation was not affected). The present demonstration of platelet aggregation by physiologically relevant concentrations of alkyl GroPCho core aldehydes suggests that alkyl GroPCho core aldehyde generated in atheroma could be involved in precipitating acute coronary events, in which thrombus formation following lipid-rich plaque disruption plays an important role.     

Differential effects of the diacylglycerol kinase inhibitor R59022 on thrombin versus collagen-induced human platelet secretion

R59022 is an inhibitor of the enzyme 1,2-diacylglycerol (DAG) kinase, which, by inhibiting the conversion of DAG to phosphatidic acid, causes an increase in endogenous DAG levels and the activity of the DAG-dependent enzyme protein kinase C. This property of the drug was utilized in the present study to assess the role of DAG, i.e., its relative importance as a potentiatory versus inhibitory mediator, in agonist-induced platelet activation. The phosphorylation of the 40-47-kDa protein by protein kinase C was monitored as an indicator of endogenous DAG levels and correlated with other agonist-induced platelet responses such as platelet aggregation, 5-hydroxytryptamine (5HT) secretion and arachidonate release, the agonists used being those that induce DAG formation, e.g., thrombin and collagen. Pretreatment of platelets with R59022 before agonist addition resulted in the potentiation of 5HT secretion as well as 45 kDa protein phosphorylation induced by thrombin and the DAG analogue, 1,2-dioctanoylglycerol (DiC8). However, collagen-induced 5HT secretion was significantly inhibited (70%) in the presence of R59022, which also had strong inhibitory effects on aggregation induced by collagen, as well as by thrombin and DiC8. The inhibition of collagen-induced secretion by R59022 was in contrast to the potentiatory effects of DiC8 on the same, suggesting that even although DAG acts as a potentiatory signal in this system, the inhibitory effects of R59022 on collagen-induced aggregation can mask any effects of endogenous DAG. This inhibitory effect of R59022 on agonist-induced platelet aggregation makes it unsuitable as a tool in studying the role of DAG in platelet activation induced by agonists such as collagen as well as the 'weak' agonists (ADP, adrenaline and platelet-activating factor), where aggregation mediates other responses such as arachidonate release and secretion. Furthermore, potentiatory effects of R59022 on 5HT secretion induced by phorbol 12-myristate 13-acetate and ionomycin, which are effects unlikely to be related to inhibition of DAG kinase was observed, and these effects further underline the non-specificity in the actions of R59022 and its limitations as a tool in studying platelet stimulus-response coupling.