Non-steroidal anti-inflammatory drugs react with two sites on platelet cyclo-oxygenase evidence from ‘in vivo’ drug interaction studies in rats

Non-steroidal anti-inflammatory drugs inhibit platelet cylco-oxygenase activity. This study shows that salicylate, diflunisal, sulphinpyrazone and indomethacine prevent in vivo aspirin inhibitory effect of cyclo-oxygenase activity as measured by the formation of malondialdehyde and thromboxane B₂, two products of platelet arachidonic acid metabolism. Salicylate also prevents the inhibitory effect of indomethacin. All these drugs therefore appear to interact with the same site on platelet cyclo-oxygenase. Since salicylate is inactive by itself on this platelet enzyme and diflunisal and sulphinpyrazone were used at ineffective doses, it is suggested that their interaction with aspirin (or indomethacin) occurs at the level of a supplementary site is necessary but not sufficient for the efficacy of these drugs as cyclo-oxygenase inhibitors. Acetylation by aspirin of the active site appears to be a phenomenon secondary to the binding of salicylate moiety to the supplementary site.     

Thrombosis is reduced by inhibition of COX-1, but unaffected by inhibition of COX-2, in an acute model of platelet activation in the mouse

Background

Clinical use of selective inhibitors of cyclooxygenase (COX)-2 appears associated with increased risk of thrombotic events. This is often hypothesised to reflect reduction in anti-thrombotic prostanoids, notably PGI₂, formed by COX-2 present within endothelial cells. However, whether COX-2 is actually expressed to any significant extent within endothelial cells is controversial. Here we have tested the effects of acute inhibition of COX on platelet reactivity using a functional in vivo approach in mice.

Methodology/Principal Findings

A non-lethal model of platelet-driven thromboembolism in the mouse was used to assess the effects of aspirin (7 days orally as control) diclofenac (1 mg.kg⁻¹, i.v.) and parecoxib (0.5 mg.kg⁻¹, i.v.) on thrombus formation induced by collagen or the thromboxane (TX) A₂-mimetic, U46619. The COX inhibitory profiles of the drugs were confirmed in mouse tissues ex vivo. Collagen and U46619 caused in vivo thrombus formation with the former, but not latter, sensitive to oral dosing with aspirin. Diclofenac inhibited COX-1 and COX-2 ex vivo and reduced thrombus formation in response to collagen, but not U46619. Parecoxib inhibited only COX-2 and had no effect upon thrombus formation caused by either agonist.

Conclusions/Significance

Inhibition of COX-1 by diclofenac or aspirin reduced thrombus formation induced by collagen, which is partly dependent upon platelet-derived TXA₂, but not that induced by U46619, which is independent of platelet TXA₂. These results are consistent with the model demonstrating the effects of COX-1 inhibition in platelets, but provide no support for the hypothesis that acute inhibition of COX-2 in the circulation increases thrombosis.     

Cathepsin G-Dependent Modulation of Platelet Thrombus Formation In Vivo by Blood Neutrophils

Neutrophils are consistently associated with arterial thrombotic morbidity in human clinical studies but the causal basis for this association is unclear. We tested the hypothesis that neutrophils modulate platelet activation and thrombus formation in vivo in a cathepsin G-dependent manner. Neutrophils enhanced aggregation of human platelets in vitro in dose-dependent fashion and this effect was diminished by pharmacologic inhibition of cathepsin G activity and knockdown of cathepsin G expression. Tail bleeding time in the mouse was prolonged by a cathepsin G inhibitor and in cathepsin G knockout mice, and formation of neutrophil-platelet conjugates in blood that was shed from transected tails was reduced in the absence of cathepsin G. Bleeding time was highly correlated with blood neutrophil count in wildtype but not cathepsin G deficient mice. In the presence of elevated blood neutrophil counts, the anti-thrombotic effect of cathepsin G inhibition was greater than that of aspirin and additive to it when administered in combination. Both pharmacologic inhibition of cathepsin G and its congenital absence prolonged the time for platelet thrombus to form in ferric chloride-injured mouse mesenteric arterioles. In a vaso-occlusive model of ischemic stroke, inhibition of cathepsin G and its congenital absence improved cerebral blood flow, reduced histologic brain injury, and improved neurobehavioral outcome. These experiments demonstrate that neutrophil cathepsin G is a physiologic modulator of platelet thrombus formation in vivo and has potential as a target for novel anti-thrombotic therapies.     

Aspirin inhibits arachidonic acid metabolism via lipoxygenase and cyclo-oxygenase in hamster isolated lungs

The effect of aspirin on the fate of exogenous arachidonic acid (AA) was investigated in isolated perfused lungs of female hamsters. During pulmonary infusion of aspirin (10 μM, 100 μM or 1 mM) 45 nmol of ¹⁴C-AA was infused in two minutes into the pulmonary circulation. The nonrecirculating perfusion effluent was collected for 6 minutes after the beginning of the AA infusion. Arachidonate infusion increased the perfusion pressure. This pressor response was completely abolished by 1 mM aspirin. When aspirin was infused into the pulmonary circulation, the amount of radioactivity was increased in the perfused lungs and decreased dose dependently in the nonrecirculating perfusion effluent. The amount of unmetabolized free arachidonate was not changed significantly by aspirin in the perfused lungs or in the perfusion effluent. In the effluent the amounts of all arachidonate metabolites, which were extracted with ethyl acetate first at pH 7.4 and then at pH 3.5 and analysed by thin layer chromatography, were decreased quite similarly by aspirin. The formation of arachidonate metabolites was completely inhibited by 1 mM aspirin. In the perfused lung tissue the amount of ¹⁴C-AA was increased by aspirin in phospholipids and neutral lipids. The present study indicates that the metabolism of arachidonic acid is inhibited by aspirin in hamster lungs not only via cyclo-oxygenase but also via other lipoxygenases.     

Controversies and future perspectives of antiplatelet therapy in secondary stroke prevention

Antiplatelet agents are a cornerstone in the treatment of acute arterial thrombotic events and in the prevention of thrombus formation. However, existing antiplatelet agents (mainly aspirin, the combination of aspirin and dipyridamole and clopidogrel) reduce the risk of vascular events only by about one quarter compared with placebo. As a consequence, more efficacious antiplatelet therapies with a reduced bleeding risk are needed. We give an overview of several new antiplatelet agents that are currently investigated in secondary stroke prevention: adenosine 5′-diphosphonate receptor antagonists, cilostazol, sarpogrelate, terutroban and SCH 530348. There are unique features in secondary stroke prevention that have to be taken into account: ischaemic stroke is a heterogeneous disease caused by multiple aetiologies and the blood–brain barrier is disturbed after stroke which may result in a higher intracerebral bleeding risk. Several small randomized trials indicated that the combination of aspirin and clopidogrel might be superior to antiplatelet monotherapy in the acute and early post-ischaemic phase. There is an ongoing debate about antiplatelet resistance. Decreasing response to aspirin is correlated independently with an increased risk of cardiovascular events. However, there is still no evidence from randomized trials linking aspirin resistance and recurrent ischaemic events. Similarly, randomized trials have not demonstrated a clinical significantly decreased antiplatelet effect by the concomitant use of clopidogrel and proton pump inhibitors. Nevertheless, a routine use of this drug combination is not recommended.     

Manipulation of thrombus formation in the hamster cheek pouch with drugs that interact with PGI2 in vitro

A single platelet thrombus was formed in an arteriole of the hamster cheek pouch by electrical stimulation followed by topical application of ADP. The sizes of the thrombi were continuously recorded with a photocell placed on a TV monitor screen and quantified by areas on the record. Repeated application of small doses of ADP (5-15 nmole/10 microliters) resulted in very reproducible formation of the thrombi, and the size of the thrombi was reduced dose-dependently by topical application of PGI2. Three drugs were tested in this model. Cyclooxygenase inhibitor (indomethacin 10 mg/kg, i.p.) increased the formation of thrombi, while a smaller dose (3 mg/kg) did not have any significant effect. This could be explained by inhibition of the generation of endogenous PGI2, since aggregation of hamster platelets by ADP was not inhibited by indomethacin in vitro. EG-626 (phthalazinol, a phosphodiesterase inhibitor) (300 mg/kg, i.p.) decreased the size of thrombus. AI-122 (1.0 mg/kg, i.p.), which has been proven to enhance PGI2 biosynthesis from isolated rat aortae, also decreased the formation. Thus, drugs such as EG-626 or AI-122 are quite promising as anti-thrombotic drugs.     

Arrestin-2 differentially regulates PAR4 and ADP receptor signaling in platelets

Arrestins can facilitate desensitization or signaling by G protein-coupled receptors (GPCR) in many cells, but their roles in platelets remain uncharacterized. Because of recent reports that arrestins can serve as scaffolds to recruit phosphatidylinositol-3 kinases (PI3K)s to GPCRs, we sought to determine whether arrestins regulate PI3K-dependent Akt signaling in platelets, with consequences for thrombosis. Co-immunoprecipitation experiments demonstrate that arrestin-2 associates with p85 PI3Kα/β subunits in thrombin-stimulated platelets, but not resting cells. The association is inhibited by inhibitors of P2Y12 and Src family kinases (SFKs). The function of arrestin-2 in platelets is agonist-specific, as PAR4-dependent Akt phosphorylation and fibrinogen binding were reduced in arrestin-2 knock-out platelets compared with WT controls, but ADP-stimulated signaling to Akt and fibrinogen binding were unaffected. ADP receptors regulate arrestin recruitment to PAR4, because co-immunoprecipitates of arrestin-2 with PAR4 are disrupted by inhibitors of P2Y1 or P2Y12. P2Y1 may regulate arrestin-2 recruitment to PAR4 through protein kinase C (PKC) activation, whereas P2Y12 directly interacts with PAR4 and therefore, may help to recruit arrestin-2 to PAR4. Finally, arrestin2(-/-) mice are less sensitive to ferric chloride-induced thrombosis than WT mice, suggesting that arrestin-2 can regulate thrombus formation in vivo. In conclusion, arrestin-2 regulates PAR4-dependent signaling pathways, but not responses to ADP alone, and contributes to thrombus formation in vivo.     

Cyclooxygenase-Independent Neuroprotective Effects of Aspirin Against Dopamine Quinone-Induced Neurotoxicity

Prostaglandin H synthase exerts not only cyclooxygenase activity but also peroxidase activity. The latter activity of the enzyme is thought to couple with oxidation of dopamine to dopamine quinone. Therefore, it has been proposed that cyclooxygenase inhibitors could suppress dopamine quinone formation. In the present study, we examined effects of various cyclooxygenase inhibitors against excess methyl L-3,4-dihydroxyphenylalanine (L-DOPA)-induced quinoprotein (protein-bound quinone) formation and neurotoxicity using dopaminergic CATH.a cells. The treatment with aspirin inhibited excess methyl L-DOPA-induced quinoprotein formation and cell death. However, acetaminophen did not show protective effects, and indomethacin and meloxicam rather aggravated these methyl L-DOPA-induced changes. Aspirin and indomethacin did not affect the level of glutathione that exerts quenching dopamine quinone in dopaminergic cells. In contrast with inhibiting effects of higher dose in the previous reports, relatively lower dose of aspirin that affected methyl L-DOPA-induced quinoprotein formation and cell death failed to prevent cyclooxygenase-induced dopamine chrome generation in cell-free system. Furthermore, aspirin but not acetaminophen or meloxicam showed direct dopamine quinone-scavenging effects in dopamine-semiquinone generating systems. The present results suggest that cyclooxygenase shows little contribution to dopamine oxidation in dopaminergic cells and that protective effects of aspirin against methyl L-DOPA-induced dopamine quinone neurotoxicity are based on its cyclooxygenase-independent property.     

Effect of drugs on urate binding to plasma proteins

The effect of various drugs on urate binding to plasma proteins was investigated in normal subjects. Whereas allopurinol, aspirin, phenylbutazone, probenecid, and sulphinpyrazone all significantly reduced plasma urate concentrations, only aspirin, phenylbutazone, and probenecid significantly impaired urate binding. Colchicine and indomethacin in the doses administered had no significant effect on plasma urate concentrations or binding. In the case of aspirin, urate binding was reduced to 25% of normal, and this effect was quickly abolished after cessation of therapy. Phenylbutazone reduced urate binding to 56% and probenecid to 46% of normal; this impairment was still detected four days after cessation of therapy. Drugs may impair urate binding by competition for plasma protein binding sites, with displacement of bound urate. Impairment of urate binding in vivo by administration of certain drugs may be relevant to the precipitation of acute gouty arthritis, to the formation of gouty tophi, and to the augmentation of uricosuria. Furthermore, the role of drugs must be seriously considered during all studies on urate binding in patients with gout.     

Comparative effects of inhibitors of arachidonic acid metabolism on erythropoiesis

The effects of a variety of inhibitors of the arachidonic acid metabolic pathway have been tested on the growth of early erythroid progenitor cell-derived colonies (CFU-E and BFU-E) in an attempt to discern whether products of the cyclo-oxygenase pathway or lipoxygenase pathway are essential for erythropoiesis. Murine erythroid progenitor cells obtained from fetal livers were cultured in the presence of erythropoietin for CFU-E and of interleukin 3 for BFU-E colony formation in response to the cyclo-oxygenase inhibitors, aspirin or sodium meclofenamate, and the lipoxygenase inhibitors, BW755C, nordihydroguiaretic acid (NDGA), phenidone, and butylated hydroxyanisole (BHA). The most potent inhibitor of colony formation (both CFU-E and BFU-E) was the selective lipoxygenase inhibitor, BW755C, followed by NDGA, phenidone and BHA. Neither aspirin nor sodium meclofenamate (10(-4) - 10(-6)M) significantly (p less than 0.05) inhibited CFU-E or BFU-E formation. These results support the hypothesis that lipoxygenase products of arachidonic acid metabolism may be essential for erythroid cell proliferation/differentiation.     

Dual role of platelet protein kinase C in thrombus formation: stimulation of pro-aggregatory and suppression of procoagulant activity in platelets

Protein kinase C (PKC) isoforms regulate many platelet responses in a still incompletely understood manner. Here we investigated the roles of PKC in the platelet reactions implicated in thrombus formation as follows: secretion aggregate formation and coagulation-stimulating activity, using inhibitors with proven activity in plasma. In human and mouse platelets, PKC regulated aggregation by mediating secretion and contributing to alphaIIbbeta3 activation. Strikingly, PKC suppressed Ca(2+) signal generation and Ca(2+)-dependent exposure of procoagulant phosphatidylserine. Furthermore, under coagulant conditions, PKC suppressed the thrombin-generating capacity of platelets. In flowing human and mouse blood, PKC contributed to platelet adhesion and controlled secretion-dependent thrombus formation, whereas it down-regulated Ca(2+) signaling and procoagulant activity. In murine platelets lacking G(q)alpha, where secretion reactions were reduced in comparison with wild type mice, PKC still positively regulated platelet aggregation and down-regulated procoagulant activity. We conclude that platelet PKC isoforms have a dual controlling role in thrombus formation as follows: (i) by mediating secretion and integrin activation required for platelet aggregation under flow, and (ii) by suppressing Ca(2+)-dependent phosphatidylserine exposure, and consequently thrombin generation and coagulation. This platelet signaling protein is the first one identified to balance the pro-aggregatory and procoagulant functions of thrombi.     

A mitogen-activated protein kinase-dependent signaling pathway in the activation of platelet integrin alpha IIbbeta3

We have recently shown that the platelet integrin alpha(IIb)beta(3) is activated by von Willebrand factor (vWF) binding to its platelet receptor, glycoprotein Ib-IX (GPIb-IX), via the protein kinase G (PKG) signaling pathway. Here we show that GPIb-IX-mediated activation of integrin alpha(IIb)beta(3) is inhibited by dominant negative mutants of Raf-1 and MEK1 in a reconstituted integrin activation model in Chinese hamster ovary (CHO) cells and that the integrin-dependent platelet aggregation induced by either vWF or low dose thrombin is inhibited by MEK inhibitors PD98059 and U0126. Thus, mitogen-activated protein kinase (MAPK) pathway is important in GPIb-IX-dependent activation of platelet integrin alpha(IIb)beta(3). Furthermore, vWF binding to GPIb-IX induces phosphorylation of Thr-202/Tyr-204 of extracellular signal-regulated kinase 2 (ERK2). GPIb-IX-induced ERK2 phosphorylation is inhibited by PKG inhibitors and enhanced by overexpression of recombinant PKG. PKG activators also induce ERK phosphorylation, indicating that activation of MAPK pathway is downstream from PKG. Thus, our data delineate a novel integrin activation pathway in which ligand binding to GPIb-IX activates PKG that stimulates MAPK pathway, leading to integrin activation.     

Manipulation of thrombus formation in the hamster cheek pouch with drugs that interact with PGI2

A single platelet thrombus was formed in an arteriole of the hamster cheek pouch by electrical stimulation followed by topical application of ADP. The sizes of the thrombi were continuously recorded with a photocell placed on a TV monitor screen and quantified by areas on the record. Repeated application of small doses of ADP (5–15 nmol/10 μl) resulted in very reproducible formation of the thrombi, and the size of the thrombi was reduced dose-dependently by topical application of PGI₂. Three drugs were tested in this model. Cycloxygenase inhibitor (indomethacin 10 mg/kg, i.p.) increased the formatiion of thrombi, while a smaller dose (3 mg/kg) did not have any significant effect. This could be explained by inhibition of the generation of endogemous PGI₂, since aggregation of hamster platelets by ADP was not inhibited by indomethacin . EG-626 (phthalazinol, a phosphodiesterase inhibitor) (300 mg/kg, i.p.) decreased the size of thrombus. AI-122 (1.0 mg/kg, i.p.) which has been proven to enhance PGI₂ biosynthesis from isolated rat aortae, also decreased the formation. Thus, drugs such as EG-626 or AI-122 are quite promising as anti-thrombic drugs.     

Synthesis of Novel 3-Butylphthalide Derivatives Containing Isopentenylphenol Moiety as Potential Antiplatelet Agents for the Treatment of Ischemic Stroke

In order to find novel antiplatelet drugs for the treatment of ischemic stroke, a series of 3-butylphthalide derivatives containing isopentenylphenol moiety were designed, synthesized and characterized with spectroscopic analyses. The in vitro antiplatelet activity results indicated that compound 3 better inhibited the arachidonic acid (AA) induced platelet aggregation than aspirin (ASP) and 3-butylphthalide (NBP). Additionally, compared with precursor NBP, compound 3 possessed outstanding antithrombotic activity in the animal experiment model, which could effectively alleviate the formation of tail thrombus and carotid artery thrombus in mice. More importantly, intraperitoneal administration of compound 3 can well protected the rats against ischemia/reperfusion-induced brain injury. Further pharmacokinetic (PK) assay indicated that compound 3 had good absorption characteristics and metabolic stability in vivo. Overall, the present research provides a new candidate compound for the treatment of ischemic stroke caused by platelet aggregation.     

The role of prostacyclin in vascular tissue.

Prostacyclin (PGI2) generated by the vascular wall is a potent vasodilator, and the most potent endogenous inhibitor of platelet aggregation so far discovered. Prostacyclin inhibits platelet aggregation by increasing cyclic AMP levels. Prostacyclin is a circulating hormone continually released by the lungs into the arterial circulation. Circulating platelets are, therefore, subjected constantly to prostacyclin stimulation and it is via this mechanism that platelet aggregability in vivo is controlled. Moreover, phosphodiesterase inhibitors such as dipyridamole or theophylline exert their antithrombotic actions by potentiating circulating prostacyclin. The prostacyclin:thromboxane A2 ratio is important in the control of thrombus formation; manipulation of this ratio by small doses of aspirin (which will inhibit mainly platelet cyclooxygenase), a selective inhibitor of thromboxane formation, or the dietary use of a fatty acid like eicosapentaenoic acid (which would be the precursor for a delta17-prostacyclin (PGI3) but is transformed by the platelets into nonaggregating thromboxane A3) might have beneficial effects as antithrombotic therapies. Prostacyclin has interesting potential for clinical application in conditions where enhanced platelet aggregation is involved or to increase biocompatibility of extracorporeal circulation systems.     

Manipulation of thrombus formation in the hamster cheek pouch with drugs that interact with PGI2in vitro

A single platelet thrombus was formed in an arteriole of the hamster cheek pouch by electrical stimulation followed by topical application of ADP. The sizes of the thrombi were continuously recorded with a photocell placed on a TV monitor screen and quantified by areas on the record. Repeated application of small doses of ADP (5–15 nmol/10 μl) resulted in very reproducible formation of the thrombi, and the size of the thrombi was reduced dose-dependently by topical application of PGI₂. Three drugs were tested in this model. Cycloxygenase inhibitor (indomethacin 10 mg/kg, i.p.) increased the formatiion of thrombi, while a smaller dose (3 mg/kg) did not have any significant effect. This could be explained by inhibition of the generation of endogemous PGI₂, since aggregation of hamster platelets by ADP was not inhibited by indomethacin $\text{in vitro}$. EG-626 (phthalazinol, a phosphodiesterase inhibitor) (300 mg/kg, i.p.) decreased the size of thrombus. AI-122 (1.0 mg/kg, i.p.) which has been proven to enhance PGI₂ biosynthesis from isolated rat aortae, also decreased the formation. Thus, drugs such as EG-626 or AI-122 are quite promising as anti-thrombic drugs.     

Inhibitors of glycoprotein biosynthesis block fertilization in the hamster

The nature and control of changes in surface carbohydrates in capacitating hamster spermatozoa were analysed by using five inhibitors of glycoprotein biosynthesis in an in vitro fertilization system. Epididymal spermatozoa were treated with amphomycin, bacitracin, tunicamycin, 2-deoxyglucose, and 2-deoxy-2-fluoro-D-glucose either during the entire period of capacitation or briefly at the end of capacitation before exposing to Con A-coated agarose beads or hamster eggs with or without their zonae pellucidae. Untreated 4½-5-hr spermatozoa exhibited nearly 100% fertilization and became bound to Con A-agarose beads mainly along the length of their flagellae, resulting in the formation of clumps on the beads. In the presence of inhibitors of glycosylation, spermatozoa did not bind to Con A-agarose beads or zona-intact oocytes and they did not fuse with the zona-free oocytes. Sperm-zona binding was also inhibited by UDP-galactose and UDP-N-acetylglucosamine, but not by UDP-glucose. Sperm motility was not damaged by these inhibitors, and zona-intact and zona-free oocytes pretreated with these inhibitors underwent normal fertilization with untreated spermatozoa. These results further strengthen the view that glycoproteins on the sperm surface may be required during different stages of fertilization, including sperm-egg fusion.     

The role of calpain in the regulation of ADAM17-dependent GPIbα ectodomain shedding

There are evidence that both a disintegrin and metalloproteinase 17 (ADAM17) and calpain are involved in platelet glycoprotein (GP)Ibα ectodomain cleavage. However, the relationship between the two enzymes in the shedding process remains unclear. Here we show that calcium ionophore A23187- and α-thrombin-induced GPIbα shedding is completely inhibited by the metalloproteinase inhibitor GM6001, whereas it is only partially inhibited by calpain inhibitors. Calpain activator dibucaine-induced GPIbα shedding was completely inhibited by both metalloproteinase and calpain inhibitors. On the other hand, calpain inhibitors did not inhibit GPIbα shedding induced by the reagents that specifically activate ADAM17. Furthermore, A23187-induced GPIbα shedding in Chinese hamster ovary cells expressing wild-type or mutant GPIb-IX was also partially inhibited by calpain inhibitors and almost completely inhibited by GM6001. Therefore, these data indicate that calpain plays an important role in the regulation of ADAM17-dependent GPIbα ectodomain shedding in both platelets and nucleated cells.     

Stimulus-response coupling in human platelets activated by monoclonal antibodies to the CD9 antigen, a 24 kDa surface-membrane glycoprotein.

The CD9 molecule is a 24 kDa surface-membrane glycoprotein present on platelets and a variety of haematopoetic and non-haematopoetic tissues. In the present study we utilized specific inhibitors of thromboxane A2 (TxA2) formation (aspirin), protein kinase C [H-7 [1-(5-isoquinolinesulphonyl)-2-methylpiperazine]] and autocrine stimulation by secreted ADP (apyrase) to modify platelet activation by a monoclonal antibody ALB-6 to the CD9 antigen. This activation is only partially inhibited by aspirin alone but, in combination with either H-7 or apyrase, more than 50% inhibition of platelet aggregation and secretion was observed. This combination of inhibitors was also required to inhibit effectively the phosphorylation of myosin light chain and the 47 kDa substrate of protein kinase C. Intracellular Ca2+ flux monitored by the fluorescent dye fura-2 showed that this was almost completely mediated by the aspirin-sensitive TxA2 pathway. We suggest that the aspirin-insensitive pathway is primarily mediated by phospholipase C formation of diacylglycerol to activate protein kinase C. The inhibition by apyrase suggests a strong dependency on autocrine stimulation by secreted ADP to fully activate both phospholipase C and express fibrinogen-binding sites mediating platelet aggregation. This alternate pathway of phospholipase C activation by ALB-6 may be mediated by cytoplasmic alkalinization [monitored by SNARF-1 (5'(6')-carboxy-10-bismethylamino-3-hydroxy-spiro-[7H- benzo[c]xanthine-1',7(3H)-isobenzofuran]-3'-one) fluorescence of the dye]. Both activation pathways are dependent on intact antibodies, since F(ab')2 fragments of SYB-1, a monoclonal antibody against the CD9 antigen with activation characteristics identical with those of ALB-6, do not elicit activation. Besides thrombin, collagen is another physiological agonist shown to induce aspirin-insensitive activation. Similarities to ALB-6 in collagen sensitivity to apyrase in combination with aspirin inhibitors were noted with respect to aggregation and secretion, as well as a complete block of Ca2+ flux by aspirin. However, it is unlikely that collagen activation is mediated by the CD9 antigen, since SYB-1 F(ab')2 fragments had no effect on collagen activation and aspirin also completely blocked the alkalinization response to collagen, in contrast with ALB-6.