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R C Carroll

Publications and source records attributed to R C Carroll.

At least 37 records · Page 2Linked to original sources
PubMed1997

Blocking platelet aggregation inhibits thromboxane A2 formation by low dose agonists but does not inhibit phosphorylation and activation of cytosolic phospholipase A2.

Inhibition of aggregation by Ro 44-9883, a potent and selective non-peptide GPIIb/IIIa antagonist, resulted in inhibition of serotonin secretion induced by weak agonists such as ADP or low doses of either thrombin receptor agonist peptide (TRAP) or collagen. In contrast, alpha granule secretion was inhibited to different extents dependent on donor, averaging 60% inhibition. Inhibition of serotonin secretion correlated with an inhibition of thromboxane A2 (TxA2) formation, both of which were overcome by higher doses of TRAP or collagen. Ro 44-9883 had no effect on the already reduced serotonin secretion and TxA2 formation in Glanzmann's thrombasthenic platelets. Restoration of serotonin secretion in the absence of aggregation requires both TxA2 and lysophosphatidic acid. In addition, Ro 44-9883 inhibition of TxA2 formation was not due to a lack of phospholipase A2 (PLA2) phosphorylation and activation as assayed in vitro. These results suggest that aggregation is required for weak or low dose agonist induced in vivo activity of PLA2, possibly by either regulating phospholipid substrate availability or interaction of PLA2 with platelet membranes.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1996

Purification of lipopolysaccharide-binding protein from bovine serum.

Lipopolysaccharide-binding protein (LBP) plays a central role in presentation of bacterial-derived lipopolysaccharide (LPS; endotoxin) to leukocytes such as macrophages and neutrophils. Interaction of LBP with LPS is significant because LBP-LPS complexes promote activation of leukocytes and the immune system, which results in enhanced secretion of a spectrum of proinflammatory cytokines. An improved, simplified method was used to purify bovine LBP from serum. Methodology consisted of ion-exchange chromatography using Bio-Rex 70 resin, followed by gel-filtration chromatography (Sephacryl S-200 resin) of a selected ion-exchange fraction (0.22-0.50 M NaCl). Densitometric scans on silver-stained polyacrylamide gels of chromatographically-derived proteins indicated up to 88.7% purity of the resultant 64kD protein (bovine LBP) in the cleanest fractions. The isoelectric point of bovine LBP was determined to be 6.8. Identity of the protein was substantiated by western-blot analysis, and by N-terminus amino acid sequence analysis with favorable comparison to published sequence data from rabbit, human, and murine LBP Identity was corroborated by use of purified bovine LBP in bioassays which demonstrated enhanced tissue factor expression of LPS (1 ng ml(-1)-stimulated bovine alveolar macrophages. Tissue factor expression was inhibitable in these assays using anti-CD14 monoclonal antibodies, which is also consistent with LBP-mediated activation of cells. When bovine LBP was heated at 56 degrees C for 30 min, the biological activity was reduced by 50% in the macrophage-based bioassays. Biological activity of bovine LBP was completely destroyed by heating at 62 degrees C for 30 min, which compared favorably with data resulting from use of fetal bovine serum.

Acute-Phase Proteins↗
PubMed1996

Soluble CD14 and lipopolysaccharide-binding protein from bovine serum enable bacterial lipopolysaccharide-mediated cytotoxicity and activation of bovine vascular endothelial cells in vitro.

Bacterial endotoxin (lipopolysaccharide, LPS) has potent proinflammatory properties toward many cell types, including vascular endothelial cells. Bovine endothelial cells are often used for investigations involving the vascular endothelium in vitro, and other bovine products such as fetal bovine serum are also widely utilized in research laboratories. Evidence is presented that soluble CD14 (sCD14) is present in bovine serum and that LPS-mediated activation and cytotoxicity to bovine endothelial cells in vitro are dependent on sCD14. LPS-mediated activation of endothelial cells was quantitated by measuring tissue factor expression using an activated factor X-related chromogenic assay. Concentrations of 0.1-5.0% fetal bovine serum in the culture medium promoted LPS-induced tissue factor expression on bovine endothelial cells, and anti-CD14 monoclonal antibody (mAb) (20 micrograms/ml) inhibited tissue factor expression, whereas control antibodies did not. LPS-mediated damage to endothelial cells was assayed using the MTT tetrazolium assay. We found that either serum or recombinant human soluble CD14 (rsCD14, 20-2000 ng/ml) was required for LPS-related endothelial cell damage and that anti-CD14 mAb inhibited cytotoxicity. In addition, bovine LPS-binding protein (LBP, 20 ng/ml) purified from bovine serum had no effect on LPS-mediated cytotoxicity, but bovine LBP greatly enhanced the cytotoxic effect of LPS plus rsCD14. Western blot analysis performed on fractionated bovine serum samples with anti-CD14 mAb revealed immunoreactivity with a 50-55-kd protein, a size consistent with sCD14. Evidence of endothelial cell-associated CD14 was not detected using an immunofluorescence technique on cell preparations, nor by Northern blot analysis. These results indicate the existence of sCD14 in bovine serum and that soluble bovine serum factors including sCD14 and LBP facilitate presentation of LPS to receptive cells.

Acute-Phase Proteins↗
PubMed1995

Coincidence detection at the level of phospholipase C activation mediated by the m4 muscarinic acetylcholine receptor.

BACKGROUND: One of the principal mechanisms by which G-protein-coupled receptors evoke cellular responses is through the activation of phospholipase C (PLC) and the subsequent release of Ca2+ from intracellular stores. Receptors that couple to pertussis toxin (PTX)-insensitive G proteins typically evoke large increases in PLC activity and intracellular Ca2+ release. In contrast, receptors that use only PTX-sensitive G proteins usually generate weak PLC-dependent responses, but efficiently regulate a second effector enzyme, adenylyl cyclase. For example, in many cell types, agonist binding by the m4 muscarinic acetylcholine receptor (m4 receptor) results in a strong inhibition of adenylyl cyclase and very little stimulation of PLC activity or release of intracellular Ca2+. We have investigated whether the weak, PTX-sensitive stimulation of PLC activity by the m4 receptor can play a significant role in the generation of cellular responses. RESULTS: We report here that PTX-sensitive Ca2+ release mediated by the m4 receptor in transfected Chinese hamster ovary cells is greatly enhanced when endogenous purinergic receptors simultaneously activate a PTX-insensitive signaling pathway. Furthermore, m4-receptor-induced transcription of the c-fos gene (a Ca(2+)-sensitive response) is similarly potentiated when purinergic receptors are coactivated. These enhanced m4-receptor-dependent Ca2+ responses do not require an influx of external Ca2+, and occur in the absence of detectable purinergic-receptor-stimulated Ca2+ release; they apparently require the activation of both PTX-sensitive and PTX-insensitive G-protein pathways. Measurements of phosphoinositide hydrolysis indicate that the enhancement of m4-receptor-mediated Ca2+ signaling by purinergic receptors is due to a synergistic increase in agonist-stimulated PLC activity. CONCLUSIONS: These studies demonstrate that the potency of m4-receptor-mediated PLC signaling is highly dependent upon the presence or absence of other PLC-activating agonists. The ability of the m4 receptor to evoke a strong, but conditional, activation of PLC may allow this type of receptor to participate in a coincidence-detection system that amplifies simultaneous PLC-activating signals through a mechanism involving crosstalk between PTX-sensitive and PTX-insensitive G-protein pathways.

Adenosine Triphosphate↗
PubMed1994

Heterotrimeric G proteins containing G alpha i3 regulate multiple effector enzymes in the same cell. Activation of phospholipases C and A2 and inhibition of adenylyl cyclase.

Previous studies have shown that a single type of transmembrane receptor is able to regulate multiple effectors through the activation of heterotrimeric G proteins. For example, the m2 muscarinic acetylcholine receptor (mAChR) expressed in Chinese hamster ovary (CHO) cells inhibits adenylyl cyclase, stimulates phospholipase C-dependent intracellular Ca2+ release, and activates phospholipase A2 through pertussis toxin-sensitive G proteins. However, it is unclear whether multiple effector enzymes can be regulated by one type of heterotrimeric G protein within a single cell. To investigate this question, we constructed a derivative of G alpha i3 (termed G alpha i3 C > S) in which the carboxyl-terminal cysteine residue, the site for pertussis toxin modification, was changed to a serine. Following pertussis toxin treatment of transfected CHO cells expressing the m2 mAChR, we found that the G alpha i3 C > S protein underwent guanine nucleotide exchange in response to the muscarinic agonist carbachol, while the m2 mAChR failed to activate the endogenous G alpha i2 and G alpha i3 proteins. Moreover, coupling of heterotrimeric G proteins containing G alpha i3 C > S to the m2 mAChR resulted in pertussis toxin-resistant inhibition of adenylyl cyclase, stimulation of phospholipase C-induced intracellular Ca2+ release, and phospholipase A2-mediated arachidonic acid release. Therefore, these studies provide conclusive evidence that heterotrimeric G proteins containing just G alpha i3 can regulate multiple effector enzymes within the same cell type.

Adenylate Cyclase Toxin↗
PubMed1994

Signal transduction pathways of bacterial lipopolysaccharide-stimulated bovine vascular endothelial cells.

Increased procoagulant activity of vascular endothelial cells may be an important component in the pathogenesis of intravascular coagulation associated with gram-negative bacterial diseases. Two bovine endothelial cell (BEC) lines isolated from pulmonary arteries (ENS-2 and ENT-18) were used in this study to investigate procoagulant signal transduction pathways of endotoxin (lipopolysaccharide, LPS)--stimulated BECs. The endothelial cell line ENS-2 was sensitive to LPS as demonstrated by tissue factor (TF) expression, but in contrast, the ENT-18 endothelial cell line was unusually resistant to the effects of LPS. No remarkable quantitative difference in binding of radiolabeled LPS was detected between the two endothelial cell lines. A protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate, PMA) failed to induce TF expression in either cell line at concentrations ranging from 0.05 to 1.00 microM when used as a sole stimulus for the endothelial cells. However, when PMA was used in combination with LPS, PMA enhanced the stimulatory effect of LPS on the endothelial cells. In parallel experiments, PKC inhibitors (H-7 and GF 109203X) interfered with the stimulatory effect of LPS on the cells by decreasing tissue factor expression. We also found that an activator of adenylate cyclase, forskolin, similarly inhibited LPS-induced tissue factor activity. In contrast, protein tyrosine kinase inhibitors (genistein, lavendustin A) had no inhibitory effect on LPS-induced endothelial cell tissue factor expression. Our results collectively suggest that activation of PKC is an important step in stimulation of endothelial cells by LPS, and that LPS and phorbol esters may synergize to produce an enhanced stimulatory effect. Our results also suggest participation of cAMP in controlling LPS-mediated stimulation of endothelial cells, but fail to demonstrate a role for protein tyrosine kinase activity.

Adenylyl Cyclases↗
PubMed1993

Transfected m2 muscarinic acetylcholine receptors couple to G alpha i2 and G alpha i3 in Chinese hamster ovary cells. Activation and desensitization of the phospholipase C signaling pathway.

Muscarinic acetylcholine receptor subtypes (m1-m5) differentially regulate phosphoinositide-specific phospholipase (PLC) through the activation of distinct guanine nucleotide-binding (G) proteins which can be distinguished on the basis of their sensitivity to inhibition by pertussis toxin (PTX). In transfected Chinese hamster ovary cells, the m2 receptor subtype regulates the stimulation of PLC and inhibition of adenylyl cyclase (AC) through PTX-sensitive G proteins. In this study, we utilized the ability of cholera toxin (CTX) to ADP-ribosylate PTX-sensitive alpha subunits as part of the ternary complex formed by heterotrimeric G proteins and agonist-bound receptors to detect and characterize the interactions between transfected m2 receptors and endogenous G proteins in Chinese hamster ovary cells. In membranes derived from cells expressing the m2, but not the m3 receptor, the cholinergic agonist carbachol stimulated CTX modification of a 40-kDa species (G alpha 40). Importantly, similar carbachol dose dependence values and PTX dose sensitivities were observed for m2 receptor-mediated PLC signaling and G alpha 40-CTX modification. High resolution urea-SDS-polyacrylamide gel electrophoresis analysis revealed that G alpha i2 (40 kDa) and G alpha i3 (41 kDa) were components of the G alpha 40 identified by m2 receptor-dependent CTX modification. Furthermore, the sensitivities of G alpha i2 and G alpha i3 to PTX modification were determined to be the same as those for PTX inhibition of G alpha 40 labeling by CTX and m2 receptor-mediated PLC signaling. Similarly, agonist-induced desensitization of m2 receptor-G protein signaling required doses of agonist associated with stimulation of PLC. Desensitization involved receptor sequestration and down-regulation of G alpha i3; however, only the reduction of G alpha i3 required prior activation PLC signaling. Finally, desensitization of m2-G protein coupling could be partially mimicked by treatment with thapsigargin, an inducer of intracellular Ca2+ release, without altering the number of cell surface receptors or G protein levels. These results demonstrate that m2 receptors couple to both G alpha i2 and G alpha i3 in vivo and that this interaction is integral to both positive and negative regulatory pathways leading to activation of PLC and desensitization of receptor signaling.

Adenylate Cyclase Toxin↗
PubMed1992

Platelet activation by cross-linking HLA class I molecules and Fc receptor.

The effect on platelet activation of monoclonal antibodies directed against common determinants of the HLA class I heavy chain molecule was studied. Cross-linking W6/32, an anti-HLA class I of IgG2a subclass, led to platelet activation. Two other antibodies of the same subclass did not have this effect on platelets. The lack of activity of the F(ab')2 fragments suggests that the activation signal is mediated by the platelet Fc receptor (Fc gamma RII). Indeed, except for a higher sensitivity of W6/32 to aspirin and apyrase, activations by cross-linking IV-3 (an anti-Fc gamma RII) and W6/32 are similar at the level of InsP3 formation, calcium mobilization, pH modifications, and activation of protein kinase C and myosin kinase. When HLA class I molecules and Fc gamma RII are cross-linked together, platelet activation occurs. This is not observed when a control IgG2a is substituted for W6/32 or when CD9 and Fc receptor are cross-linked together. This suggests that HLA class I molecules and Fc gamma RII synergize to activate platelets.

Adenosine Diphosphate↗
PubMed1992

Low concentrations of sodium azide specifically inhibit a thromboxane A2 pathway in human platelets.

Sodium azide completely inhibits the serotonin release induced by ADP, arachidonic acid and the thromboxane A2 mimetic U46619, but does not have any effect on the activation by PMA. Collagen and thrombin are inhibited when used at low concentrations, but not at high concentration. This pattern of activation suggests that the inhibition by azide is not a metabolic inhibition. The antagonism of U46619-induced secretion was further studied and was shown to be non-competitive. It is selective for certain components of the U46619 stimulus-response coupling: aggregation, serotonin secretion and the activation of protein kinase C are completely or almost completely inhibited by 300 microM sodium azide. Shape change, calcium elevation, cytoplasmic alkalinization and phosphorylation of myosin light chain are only partially modified. This suggests that azide may specifically inhibit one of the different forms of thromboxane A2 receptors present in platelets.

Adenosine Diphosphate↗
PubMed1991

Fc gamma receptor-mediated interplatelet activation by a monoclonal antibody against beta 2 microglobulin.

Three different mAb directed against beta 2 microglobulin (two IgG1 and one IgG2a) were tested for their ability to activate human platelets. Although all three antibodies bound to platelets, only one of them, B2.62.2, of the IgG1 subclass, induced platelet activation. This activation is similar to the activation by SYB-1, a CD9 antibody of the same subclass previously described as activating platelets through platelet Fc gamma R. These similarities include serotonin secretion, a lag time preceding aggregation and the induction of a strong intracellular calcium mobilization from storage pools. As with CD9 antibodies, the F(ab')2 fragments of B2.62.2 did not induce activation but blocked the activation by the native antibody, by preventing the binding to beta 2 microglobulin. Also, this activation was inhibited by pretreating the platelet with IV-3, a mAb that blocks the Fc binding site of the FcR. Inasmuch as the same antibody does not prevent the binding of B2.62.2 on platelets, we conclude that the activation by B2.62.2 is mediated by the FcR. Nevertheless, there were differences with the activation by SYB-1. B2.62.2 activation was more dependent on thromboxane A2 formation and no cytoplasmic alkalinization was detected. Finally, contrary to SYB-1, B2.62.2 activation proved to be sensitive to platelet count, suggesting that it involves the formation of immune complexes consisting of antibodies and platelets, that activate nearby platelets.

Antibodies, Monoclonal↗
PubMed1991

Interaction of two GPIIb/IIIa monoclonal antibodies with platelet Fc receptor (Fc gamma RII).

We have previously used the IV-3 monoclonal antibody specific for Fc gamma RII to demonstrate that platelet activation by CD9 monoclonal antibodies such as ALB-6 is mediated by the Fc gamma RII. Here, we show that platelet activation following addition of a monoclonal antibody directed against GPIIb/IIIa, P256 is completely blocked by IV-3, as monitored by serotonin release, calcium and pH modifications. However, aggregation was only partially inhibited. D3GP3 is another monoclonal antibody directed against GPIIIa which has been shown to induce platelet aggregation by exposure of the fibrinogen binding site. The present study demonstrates that this phenomenon is not accompanied by calcium flux or pH modification, nor is it blocked by pretreatment of platelet by IV-3. Despite its apparent independence from the Fc gamma RII activation pathway, D3GP3, but not its Fab fragment, was able to inhibit ALB-6 induced activation, including serotonin release, calcium flux and pH modifications. Binding studies demonstrated that D3GP3 (20 micrograms/ml, 0.13 microM) does not block ALB-6 binding to CD9 antigen but completely blocks IV-3 binding to the Fc receptor for concentrations of IV-3 ranging from 0 to 15 nM. Together, these results suggest an interaction between GPIIb/IIIa, Fc gamma RII and GPIIb/IIIa monoclonal antibodies which in some cases can result in activation of platelets through Fc gamma RII.

Antibodies, Monoclonal↗
PubMed1991

Platelet aggregation, storage pool deficiency, and protein phosphorylation in mice with Chediak-Higashi syndrome.

The beige (bgJ/bgJ) mouse is a well-described murine model of Chediak-Higashi syndrome. Platelet function was examined in normal and beige mice to better characterize the defective aggregation response in platelets from mice with Chediak-Higashi syndrome. Platelet aggregation after collagen, thrombin, and phorbol-12-myristate 13-acetate stimulation was significantly (P less than 0.025) decreased in platelets from beige mice, relative to platelets from normal mice. Compared with beige and normal mice, those heterozygous for the bg trait had intermediate responses to collagen and thrombin, but not phorbol-12-myristate 13-acetate. The defect(s) in aggregation of platelets from beige mice was associated with a dense granule storage pool deficiency and decreased stores of serotonin and adenine nucleotides in platelets. Mice heterozygous for the bg trait had normal platelet serotonin and adenine nucleotide concentrations. Platelets from beige mice were approximately 10 times more sensitive to prostacyclin inhibition of collagen-induced aggregation than were platelets from control mice. However, a significant difference in platelet cyclic AMP concentration was not apparent between beige and normal mice after prostacyclin stimulation. Platelet endoperoxide synthesis measured by quantification of thromboxane B2, was normal in beige mice. Protein phosphorylation patterns in mouse platelets were similar to those seen in human platelets. Thrombin and collagen-induced [32P] phosphorylation of 40- and 20-kD proteins in platelets from normal and beige mice was similar. Results indicate that the biochemical defect(s) in platelet function in beige mice is partially attributable to storage pool deficiency and does not result in an absolute defect in phosphorylation of 40- and 20-kD proteins.

Animals↗
PubMed1991

Characterization of platelet function in cyclic hematopoietic dogs.

Platelet aggregation to incremental doses of eight different platelet agonists (collagen, thrombin, platelet-activating factor [PAF], arachidonic acid [AA] plus epinephrine, the calcium ionophore A23187, ADP, phospholipase C [PLC], and 12-O-tetradecanoyl phorbol-13-acetate [TPA]) was compared in normal (N) and cyclic hematopoietic (CH) dogs. Platelet aggregation was defective with collagen, PAF, TPA, and possibly thrombin as agonists but normal when ADP, PLC, arachidonic acid plus epinephrine, and A23187 were used as agonists with CH platelets. In heterozygous CH dogs, platelet aggregation was intermediately defective when tested with collagen and PAF as agonists. Thromboxane B2 (TXB2) concentrations (mean +/- SD; pg/10(6) platelets), as measured by RIA, were similar in CH and normal dogs both prior to (CH: 7.6 +/- 7.0; N: 5.5 +/- 3.9) and after collagen stimulation (collagen: 141.3 +/- 42.5; 123.1 +/- 38.4). Granule storage pools of serotonin and platelet adenine nucleotides were markedly decreased in homozygous CH but not heterozygous CH dogs. Thrombin stimulated phosphorylation of 40- and 20-kd proteins in platelets from CH and normal dogs to an equal extent. However, collagen-stimulated phosphorylation of the 40- but not the 20-kd protein was significantly decreased in platelets from CH dogs. These data suggest that there is a biochemical defect in platelets from CH dogs that results in storage pool disease and decreased phosphorylation of a 40-kd protein.

Animals↗
PubMed1990

Extensive C1q-complement initiated lysis of human platelets by IgG subclass murine monoclonal antibodies to the CD9 antigen.

Several monoclonal antibodies (MAbs) to CD9, a surface membrane glycoprotein of 24 kDa found on platelets as well as several other hematopoietic and nonhematopoietic tissues, have the property of activating platelets. We have recently shown that with two of these MAbs (ALB-6 and SYB-1) this activation is mediated by interaction of the Fc portion of these IgG1 subclass MAbs with the Fc gamma II receptor (FcRII) and is blocked by a MAb to this receptor (IV-3). In this report we show that several MAbs to the CD9 antigen (BA-2, BU-16, MM2/57) also cause extensive and rapid platelet lysis in plasma. This lysis is mediated by the classical complement pathway dependent on C1q fixation. Lysis was not blocked by inhibiting platelet activation with prostaglandin E1 or by the MAb to FcRII (IV-3). The CD9 MAb BU-16 can also activate platelets through the FcRII when complement lysis is prevented by removal of C1q using specific antisera or by isolation of the platelets from plasma.

Animals↗
PubMed1990

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.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1990

Platelet activation by CD9 monoclonal antibodies is mediated by the Fc gamma II receptor.

The function of the human cell surface CD9 antigen is not known, yet monoclonal antibodies (mAbs) of the IgG1 subclass in the CD9 cluster induce activation of platelets. Previously it had been shown that this activation pathway is comparable both in kinetics and extent to physiological agonists such as thrombin. Here it is demonstrated that activation with CD9 mAbs depends on interaction of the Fc part of the CD9 antibody molecule with Fc receptors on the platelet surface, since: (i) mAb directed against the Fc receptor totally blocked the platelet response to CD9 mAb; and (ii) F(ab')2 fragments of the CD9 mAb SYB-1 which bound to platelets, as demonstrated by flow cytometry, failed to activate them. Furthermore, platelet activation by CD9 mAb closely paralleled the activation caused by cross-linking Fc receptors when comparing: (i) kinetics and extent of aggregation; (ii) thromboxane synthesis; (iii) calcium flux; and (iv) the cytoplasmic alkalinization response. Thus it is concluded that CD9 antigen itself does not necessarily participate in stimulus-response coupling leading to platelet activation by CD9 mAbs, and that this activation can be entirely accounted for by the Fc receptor pathway mechanism. The results suggest a possible novel mechanism for platelet consumption in cases of immune thrombocytopenia.

Antibodies, Monoclonal↗