Exposure Of Fibrinogen Receptors Research Articles

P151 Characterization of platelet activation profile in Inflammatory Bowel Disease

Abstract Background Platelet-related markers associated with a procoagulant state have been described for Immune-mediated Inflammatory Diseases (IMIDs), but a broader profiling of these abnormalities in Inflammatory Bowel Disease (IBD) is lacking. Methods Cross-sectional study to describe and compare platelet biomarkers of IBD outpatients, both Crohn’s Disease (CD) and Ulcerative Colitis (UC), vs healthy controls (HC). Patients over 18 years old with a diagnosis of CD or UC according to the European Crohn’s and Colitis Organization (ECCO) criteria were included. Fecal calproctectin (Fcal) >=150ug/g was used as a cutoff level for active disease. Exclusion criteria for IBD patients are listed under Table 1. Fibrinogen (FBG) receptor on platelet’s surface was determined with anti-CD41 and anti-CD61 monoclonal antibodies (mAbs); and von Willebrand factor receptor with anti-CD42a and anti-CD42b mAbs. Platelet activation markers [TRAP and ADP-induced activation of fibrinogen (FBG) receptor, and P-selectin (Psel) and CD63 exposure] were determined, respectively, through binding of PAC1, anti-P-selectin and anti-CD63 (mAbs). All variables were determined by flow cytometry. Results 55 patients (27 CD, 28 UC) and 75 HC have been included. 43 IBD patients had active disease (21 CD, 22 UC). Complete baseline characteristics of the IBD population are described in Table 1. Basal Psel (% of positive cells) was increased in CD vs UC (median [IQR]: 5.64 [4,4-10,4] vs 2,9 [1,8-6,8], p=0,02) and vs HC (3,5 [1,4-6,8], p=0,008). ADP-induced Psel was increased in CD vs HC (mean, SD: 49.92, SD 12.75 vs 41.943, SD13.15, p=0,007). Basal CD63 (% of positive cells) was significantly higher in active IBD vs HC (W = 1143, p = 0.02) and inactive IBD vs HC (W = 528, p= 0.006). CD42a (% of positive cells) was decreased in IBD vs HC (W = 1128.00, p=0.007) and CD vs HC (W = 777.00, p =0.04). CD42b (% of positive cells) was also decreased in IBD vs HC (mean, SD: 106.9 SD, 17.32 vs 115.7, SD 28.42, p=0,01) and CD vs HC (104.06 SD 14.49, p=0,03). No differences in basal or TRAP/ADP induced PAC binding, and in CD41 or CD61 expression were observed among groups. Multiple linear regression showed association of Fcal with basal Psel in IBD patients (p=0,02); and Spearman’s correlation showed negative moderate correlation between Fcal and basal Psel (r=0,3; p=0,003) Conclusion Features of platelets in IBD suggest increased platelet degranulation and reduced vWF receptor with regards to healthy controls, specially in CD, but no differences in FBG-related receptors. Inverse moderate correlation between Fcal and Psel in IBD patients suggests depletion of intra-platelet granules at higher levels of gut inflammation.

Unexpected low expression of platelet fibrinogen receptor in patients with chronic myeloproliferative neoplasms: how does it change with aspirin?

SummaryThis study was conducted to evaluate the expression of fibrinogen receptors on platelets of Philadelphia‐negative chronic myeloproliferative neoplasm (MPN) patients. We collected blood samples from 40 consecutive MPN patients and healthy volunteers. We performed flow cytometry analysis of P‐selectin expression and integrin beta‐3, activation of glycoprotein (GP) IIb/IIIa and fibrinogen receptor exposure (PAC‐1 binding). Surprisingly, we found a very low PAC‐1 binding capacity in MPN patients; however, the expression of PAC‐1 was almost completely recovered with aspirin intake. We hypothesize that the hypercoagulable states observed in MPN patients could depend on a primarily plasma‐driven impairment of fibrin turnover and thrombin generation.

Platelet Dysfunction and Cellular Microparticles May be Involved in the Hipercoagulable State Observed in Obstructive Sleep Apnea Syndrome

Introduction: Obstructive sleep apnea syndrome (OSA) is a common disorder characterized by repetitive episodes of nocturnal breathing cessation due to upper airway collapse that cause intermittent hypoxia. Because of this, there is an increase in oxidative stress, endothelial damage and mortality associated with the incidence of thrombotic events. The evaluation of platelet function by flow cytometry (FCM) and hemostatic capacity through global hemostasis tests have been successfully used in the study of the prothrombotic state associated to numerous diseases. We anticipate that these techniques may help to clarify the mechanism involved in the prothrombotic state of OSA.Materials and Methods: We included 16 OSA patients diagnosed in the Pneumology Unit of the University Hospital La Paz and 59 healthy controls recruited in the Donor Unit of the same Hospital.Platelet surface receptors were analysed by FCM (FACScan, BD Biosciences) with specific monoclonal antibodies (mAbs) against CD41/αIIb and CD61/β3, CD42a and CD42b. Platelet activation was assessed by FCM through PAC1-FITC binding (mAbs that recognizes activated conformation of fibrinogen receptor), P-selectin and CD63 exposure in baseline condition and after stimulation with 100 µM TRAP (agonist of the PAR-1 receptor) and 10 µM ADP. Platelet apoptosis was analysed by FCM through FITC-annexin V (BD Pharmingen) binding to phosphatidylserine (PS) exposed on platelet membrane under basal conditions and by determination of activity of caspases -3/7, -8, and -9 (BD Millipore).The overall hemostatic capacity was determined by the evaluation of thrombin generation using the Calibrated Automatic Thrombinography (CAT) with fresh platelet-poor plasma (obtained after double centrifugation of whole blood at 2500g, 20 minutes and 24ºC) and by the assessment of kinetics of clot formation using Rotational Thromboelastometry (ROTEM® Gamma, Tem Innovations GmbH, Spain) using whole blood and activation by recalcification (naTEM® test, Tem Innovations GmbH, Spain).The statistical analysis was performed using SPSS 9.0 software (SPSS Inc., Chicago, Illinois, USA). Statistical significance was established at p <0.05.Results: No significant differences in age or sex were found between groups. Patients presented anxiety-depressive disorder (15%), ischemic cardiomyopathy (8%), dyslipidemias (38%), arterial hypertension (69%) and lower minimum oxygen pressures during sleep (mean±SD; patients: 76±8 % vs controls: 89±1 %; p< 0.001) indicating hypoxia. In basal conditions, an increase of CD42a subunit of the von Willebrand factor receptor was observed in patients with OSA but no differences were found in the exposure of fibrinogen receptor either at basal condition or after activation with TRAP or ADP (Table 1 and Figure 1). Platelet PS exposure before and after TRAP stimulation was higher in the patient group (Figure 1) but no differences between groups were observed in caspase activities (Table 2). Clotting time (CT), maximum clot firmness (MCF) and MP-associated thrombin generation were higher in patients with OSA (Figure 2).Conclusion: Mechanisms related to the mobilization of P-selectin to platelet surface and with the maintenance of phospholipids distribution in platelet membrane might be altered in OSA. The increment of P-selectin exposure may favor the interaction between white cells and platelets through PSGL-1 ligand from lymphocytes which in turn may produce endothelial damage explaining part of OSA's pathophysiology. A lower CT and higher MCF reflected a hypercoagulable state in patients, perhaps related to their higher MP-associated thrombin generation and, at least in part, due to the enhanced platelet-PS exposure. Our results also suggest that both ROTEM and CAT are useful tools for characterizing prothrombotic states in OSA patients.Work supported by grant from FIS-FEDER PI15/01457. NB holds a Miguel Servet II (FIS-FEDER CP14/00024). DisclosuresÁlvarez-Roman:Shire: Consultancy; NovoNordisk: Consultancy; SOBI: Consultancy. Jimenez-Yuste:Grifols: Consultancy, Research Funding; Octapharma: Consultancy, Research Funding; CSL Behring: Consultancy; Bayer: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Shire: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; NovoNordisk: Consultancy, Research Funding; Sobi: Consultancy, Research Funding. Butta:FIS-Fondos FEDER: Research Funding.

Unexpected Low Expression of Platelet Fibrinogen Receptor in Patients with Chronic Myeloproliferative Neoplasms: How Does It Change with Aspirin?

INTRODUCTIONPatients affected by Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) are considered at high risk of thrombo-haemorrhagic events, but the role of the platelet count in the assessment of the risk of vascular events is still controversial. A tight correlation was found between the platelet count and plasma sCD40L, which appears to be required for thrombus formation in vivo. However sCD40L is increased both in MPNs and reactive thrombocytosis. Intravascular aggregates of platelets and leukocytes, mediated by P-selectin and CD11b on the former and the latter, respectively, have been observed.Both of this processes should imply a perpetual - and measurable - platelet activation. Several studies on platelet function have been already proposed, nevertheless the mechanisms through which platelets are able to trigger vascular events, are not yet adequately clarified.A refined method for the determination of platelet activation appears to be the use of platelet PAC-1 antibody, able to identify the expression of the fibrinogen receptor of platelet glycoprotein IIb/IIIa. This expression is indeed unique in the process of platelet activation, and yet rarely analyzed. Moreover, since the platelet fibrinogen receptor exposure seems to be influenced by turbulence in blood flow, we have thought that its evaluation could provide important biological evidences to explain some clinical manifestations, such as microvascular disturbances.METHODSBlood samples from 40 consecutive MPNs patients who never received cytoreductive agents, were obtained. 28/40 patients were receiving a continuative antiplatelet prophylaxis with low dose aspirin (ASA, 75-100 mg) at the time of collection, while 12/40 of them were not on such therapy. Our aim was to verify the expression of platelet fibrinogen receptors (PFRs) in the two different groups of patients compared to healthy volunteers, using whole blood flow cytometry. In each experiment sodium citrate and heparin (positive control of platelet activation) tubes were collected from the same patient. Within 10 minutes from blood sampling, 5 ml of whole blood from each tube was incubated for 20 minutes at room temperature in the dark with saturating concentration of CD61 PerCP, CD62P PE and PAC-1 FITC. Positive control was also incubated with PAC-1 in the presence of Arg-Gly-Asp-Ser (RGDS) in order to test the specific antibody binding. Samples were fixed with paraformaldehyde 1% for 30 minutes at 4°C in the dark and analyzed on a flow cytometer.RESULTSSurprisingly, we have been able to verify a very low PAC-1 binding to platelets in patients with MPNs not receiving cytoreduction nor antiplatelet agents (33%) if compared to that observed in healthy subjects (61%; p<0.0001). The use of aspirin seems conversely to restore the expression of platelet fibrinogen receptor, as PAC-1 binding capacity is comparable to that of healthy volunteers (56%). No difference was found with respect to the JAK2 Val617Phe mutation and its allele burden. Interestingly, by focusing on the group of patients under antiplatelet prophylaxis and with no history of thrombosis, it was found that subjects with persistent microcirculatory disorders show a higher PAC-1 binding capacity if compared to the asymptomatic ones (67% vs 52%, p= 0.04).CONCLUSIONIn untreated MPNs, a large amount of platelets are resting in a conformation that is unable to bind fibrinogen, as demonstrated by the low PAC-1 expression in cytofluorimetry. This lack of activity can be reversed by administering ASA, which is also known for its fibrinolytic and hypoprothrombinemic effects. We hypotesize that the hypercoagulable states observed in these patient could depend on a primarly plasma-driven impairment of fibrin turnover and thrombin generation. Further investigations are going to be promoted by our Group. PAC-1 could be at the same time a good marker of aspirin resistance in patients experiencing microcirculatory disorders. [Display omitted] DisclosuresMartinelli:Janssen: Consultancy; Jazz Pharmaceuticals: Consultancy; Ariad/Incyte: Consultancy; Pfizer: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy; Abbvie: Consultancy; Roche: Consultancy; Novartis: Speakers Bureau.

Polo-Like Kinase 3, a Novel Regulator of Thrombosis: Positive Regulation of TxA2 Generation, Granular Secretion and Clot Retraction, but Negative Regulation of Integrin Activation

AbstractAbstract 379Polo-like kinase (Plk) family members are serine/threonine kinases involved in cell cycle regulation. Their expression and function in platelets are not known. We identified the presence of Plk3, a member of this family, in human and mouse platelets. We found that Plk3 is localized to the filopodia of activated platelets. Furthermore, it co-immunoprecipitates with integrin aIIbb3 in an aggregation-dependent manner. To understand the physiological function of Plk3 in thrombosis, we obtained Plk3−/− mice in C57Bl/6 background, examined the tail bleeding time of Plk3−/− mice, and compared it to the Plk3+/+ (WT) mice of the same genetic background. We found that the average tail bleeding time for WT mice was about 130 s, consistent with the values reported in the literature. Interestingly, the Plk3−/− mice had a significantly (P<0.05) delayed average tail bleeding time (325 s), suggesting that Plk3 deficiency results in a bleeding phenotype. These results suggest that there may be defects in the thrombotic process in these mice. To evaluate the in vivo thrombotic phenotype, we performed a 10% FeCl3-induced carotid artery injury and observed any differences in time of occlusion or unstable occlusions in Plk3−/− mice compared to WT mice. Consistent with our finding of extended tail bleeding time in Plk3−/− mice, our results showed that the WT mouse vessel occluded within 7–9 min, whereas Plk3−/− mouse took nearly twice that time (∼14 min) to initiate vessel occlusion (P<0.001). We also performed a collagen/epinephrine-induced pulmonary thromboembolism assay to investigate the role of platelet Plk3 in thrombosis. Our result suggests a marked protection from thromboembolism in Plk3−/− mice, since significantly more (P<0.0004) survived compared to WT mice. By assessing the ability of Evans blue dye to pass through the pulmonary circulation, we determined that this better survival rate in Plk3−/− mice is due to the failure of occlusion of pulmonary vessels in these mice. This was further supported by the histological examination of the lungs of these mice, which showed decreased size of the emboli and the reduced extent of arterial occlusion compared to WT. Ex vivo platelet functional studies suggested that thrombin-induced generation of TxA2, a potent activator of platelet function, was significantly attenuated (P<0.03) in Plk3−/− mice compared to WT. When tested for activation of cPLA2, a key enzyme in TxA2 generation, we found that the phosphorylation of cPLA2 is significantly attenuated (P<0.05) in Plk3 null platelets. Furthermore, thrombin-induced secretion of both a- and d- granules was significantly reduced (P<0.007) in Plk3−/− mouse platelets compared to WT, consistent with the observed anti-thrombotic phenotype in vivo. Surprisingly, however, platelet aggregation by low dose of thrombin or PAR4 peptide was significantly augmented (P<0.02) in Plk3 null platelets compared to WT. This was further supported by the significantly increased (P<0.05) fibrinogen receptor exposure on platelets. To determine the molecular mechanism of the observed hyperaggregation, we analyzed signaling events such as ERK1/2 and Akt, an upstream regulator of integrin aIIbb3 activation. Interestingly, we found that agonist-induced activation of ERK2 and Akt (both T308 and S473 phosphorylation) is significantly enhanced in the absence of Plk3. Furthermore, we found that levels of PTEN, a negative regulator of PI3-K/Akt pathway is reduced in the absence of Plk3. The severity of the anti-thrombotic phenotype in Plk3−/− mice may have been dampened due to the opposing role of Plk3. We next asked if the integrin outside-in signaling is also enhanced in these mice. As expected, platelet adhesion to immobilized fibrinogen was significantly increased (P<0.05) in the absence of Plk3. Surprisingly, when analyzed for fibrin clot retraction, we found that Plk3 null platelets failed to retract fibrin clot. These results suggest that Plk3, a mitotic kinase, plays a significant role in regulation of platelet function such as TxA2 generation, granular secretion, and clot retraction, thus affecting the process of thrombosis. Disclosures:No relevant conflicts of interest to declare.

Two Regions of the Human Platelet F11-Receptor (F11R) Are Critical for Platelet Aggregation, Potentiation and Adhesion

The F11 receptor (F11R) was first identified on the surface of human platelets as a target for a stimulatory monoclonal antibody (M.Ab.F11) that induces secretion, followed by exposure of fibrinogen receptors and aggregation. Cloning of the gene of F11R has revealed that this protein is a cell adhesion molecule (CAM), a member of the Ig superfamily and an ortholog of the murine protein called junctional adhesion molecule (JAM). The present study has identified two domains through which M.Ab.F11 triggers a platelet response culminating with aggregation. M.Ab.F11-mediated platelet adhesion, and the potentiation of collagen and ADP-induced platelet aggregation by M.Ab.F11, were found to involve the same two domains. A F11R recombinant protein (sF11R) completely inhibited platelet aggregation, adhesion and potentiation induced by M.Ab.F11, indicative that the active conformation of the external domain of F11R is present in the soluble, secreted recombinant protein. Furthermore, a specific peptide containing the sequence of the N-terminal amino acids S-1 to C-23 of F11R, and a peptide with the sequence of K-70 to C-82 in the 1st immunoglobulin-like (Ig) fold of F11R, both inhibited M.Ab.F11-induced aggregation, adhesion and potentiation of the aggregation of human platelets. Modeling of the 3D structure of the extracellular domain of the human platelet F11R suggests that these two regions form an active site within the conformation of this CAM. The sequence of these functional domains of F11R (in the N-terminus and 1st Ig-fold) provide the basis for new drug development in the treatment of certain types of thrombocytopenia and inflammatory thrombosis.

Concomitant activation of G i protein-coupled receptor and protein kinase C or phospholipase C is required for platelet aggregation

It has recently been suggested that the concomitant activation of two distinct G protein-coupled receptors (G i and G q) is essential for platelet aggregation: in fact, the thromboxane A 2 synthetic agonist, U46619, which causes the selective activation of G q, is not able to elicit fibrinogen receptor exposure unless ADP or epinephrine is present. In the present study we demonstrate that a direct G q activation is not required for platelet aggregation and that the activation of an enzyme downstream of G q, such as phospholipase C (PLC) or protein-kinase C (PKC), is sufficient for such a process. In fact, platelet aggregation occurred in response to the snake venom toxin convulxin, which activates the PLC isoform PLCγ2 or to cytosolic PKC activator phorbol 12-myristate 13-acetate (PMA) provided a G i protein-coupled receptor was activated by ADP or epinephrine. The evidence that the PKC inhibitor, Ro 31-8220 did not suppress platelet aggregation in response to convulxin plus ADP or epinephrine led us to conclude that PLC and PKC are both involved in platelet aggregation, although not concomitantly, provided a G i protein-coupled receptor is activated.

Reversible Exposure of Human Platelet Fibrinogen Receptors by Antiplatelet Tetraspanin Monoclonal Antibodies via Induction of a Conformational Change in Membrane Glycoprotein IIb/IIIa Complex

Antihuman platelet tetraspanin (CD9 antigen) monoclonal antibodies, HI117 and SJ9A4, can induce human platelet aggregation and secretion. As platelet aggregation is mediated by fibrinogen binding to its receptors exposed on platelet glycoprotein IIb/IIIa complex, we, therefore, investigated the induction of platelet fibrinogen receptors by HI117 and SJ9A4. It was found that HI117 and SJ9A4 induced specific fibrinogen binding to human platelets, suggesting that the two monoclonal antibodies evoked obvious exposure of fibrinogen receptors on human platelets. But in the absence of fibrinogen, the monoclonal antibody-exposed fibrinogen receptors gradually lost their capacity to bind fibrinogen and closed. Our results also showed that HI117 and SJ9A4, when activating platelets, caused a conformational change in glycoprotein IIb/IIIa complex, which must contribute to the exposure of functional fibrinogen receptors on this integrin. The effect of HI117 and SJ9A4 on glycoprotein IIb/IIIa complex seems, however, to be indirect, because the HI117 and SJ9A4-induced fibrinogen binding was reduced by pretreatment of platelets with sphingosine, aspirin, apyrase, and/or PGI2. Taken together, we conclude that the antihuman platelet tetraspanin monoclonal antibodies, HI117 and SJ9A4, reversibly expose platelet fibrinogen receptors via inducing a conformational change in glycoprotein IIb/IIIa complex. Three signaling pathways, that is, thromboxane, secreted ADP, and cAMP pathways may be involved in this process, while protein kinase C activation seems to be the final common step of the three pathways.

Platelet protective effect of TAK-029, a novel glycoprotein IIb/IIIa antagonist: an in vitro study.

Previous studies have indicated that exposure of fibrinogen receptors associated with the glycoprotein IIb/IIIa complex contributes to platelet loss during cardiopulmonary bypass. TAK-029 is a newly developed reversible, nonpeptide inhibitor of platelet glycoprotein IIb/IIIa receptors. In this study, we tested the platelet preserving effect of TAK-029 in an in vitro model. The methods included the comparison of the release of beta-thromboglobulin (beta-TG) between a TAK-029 group (n = 5) and a control group (n = 5) in a mock circulation under a shear force generated by a centrifugal pump. To evaluate the degree of beta-TG release, deltabeta-TG/deltaT was calculated where deltabeta-TG is the increase in beta-TG and deltaT is the time. The results showed that the value of deltabeta-TG/deltaT in the TAK-029 group was significantly lower than it was in the control group (4.22 +/- 0.27 x 10(2) ng/ml vs. 7.33 +/- 0.66 x 10(2) ng/ml, respectively). In conclusion, TAK-029 reduced the platelet activation under the shear forces of an in vitro model, suggesting that TAK-029 is a potential candidate for platelet protection during cardiopulmonary bypass.

Lorenzo's oil and platelet activation in adrenomyeloneuropathy and asymptomatic X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is an inherited disorder of peroxisomal beta -oxidation, which results in accumulation of very long-chain fatty acids, causing damage to the nervous system, adrenal cortex and testis. The two most frequent phenotypesare childhood cerebral adrenoleukodystrophy (CCALD) and adrenomyeloneuropathy (AMN). Some affected males demonstrate no clinical signs (asymptomatic ALD), whereas female carriers can also be affected. Patients with X-ALD have been treated with Lorenzo's oil, a 4:1 combination of oleic acid and erucic acid, with thrombocytopenia as the main side effect and sometimes leading to a hemorrhagic diathesis. We studied platelet count, size and membrane surface exposure of platelet activation antigens in 17 adult X-ALD patients. Eight patients used the prescribed amount of erucic acid (as glyceroltrierucate) or more (very compliant), five used less(compliant), and four did not use the diet. All eight very compliant patientshad highly enlarged platelets and seven manifested thrombocytopenia. An enhanced in vivo platelet activation status was established by increased platelet surface expression of P-selectin (CD62P, PADGEM, GMP-140) in five of the seven thrombocytopenic patients, and of increased fibrinogen receptor exposure (measured with the antibody PAC-1) in three of these five patients. The other nine compliant or untreated patients had normal platelet counts and, generally, normal P-selection and fibrinogen receptor expression. A diet-induced 7- to 27-fold enrichment of erucic acid was observed in the platelets of the four patients studied. We conclude that the thrombocytopenia in AMN patients using Lorenzo'soil is associated with circulating platelets that have an increased erucic acid content, size and activation status. We hypothesize that the erucic acid in some way induces the increased size and thus, directly or indirectly, increased platelet activation or instability in vivo . This then causes the thrombocytopenia, with circulating platelets representing a population that has not yet been sufficiently changed to be removed, but has clear signs of activation.

Platelet membrane fluidity and intraplatelet Ca2+ mobilization are affected in uraemia.

In present investigations, platelet membrane fluidity and intraplatelet Ca2+ mobilization were analysed in uraemic platelets by fluorescence techniques. Thirteen non-dialyzed uraemic patients and 16 control subjects were examined. Anisotropy of DPH-probe, measured at 37 degrees C, was significantly higher in control (0.2236 +/- 0.0050) than in uraemic platelets (0.1969 +/- 0.0082; p < 0.01). There was no difference between control (109.8 +/- 6.0 nM) and uraemic platelets (100.0 +/- 7.3 nM) when the basal [Ca2+]i in resting platelets was determined. Activation of platelets by ADP (12.5 microM) or by thrombin (0.1 U/ml) resulted in an increase in [Ca2+]i. It was significantly higher (p* < 0.003 for ADP and p* < 0.009 for thrombin, respectively) in control platelets (383.6 +/- 56.3 nM and 2031.0 +/- 298.8 nM, respectively) than in uraemic ones (191.0 +/- 21.3 nM and 838.7 +/- 144.1 nM, respectively). The amount of released Ca2+ was higher in control platelets activated by both ADP and thrombin (157.6 +/- 21.4 nM and 409.3 +/- 71.0 nM, respectively) than in uraemic platelets (76.7 +/- 15.7 nM and 203.0 +/- 29.3 nM, respectively) and the differences were significant (p < 0.01 and p* < 0.01, respectively). These results indicate an abnormal intracellular Ca2+ mobilization in uraemic platelets. Both increased membrane fluidity and decreased Ca2+ mobilization should be considered as a possible reason of reduced fibrinogen receptor exposure on uraemic platelets.

Signaling through G Proteins in Platelets: to the Integrins and Beyond

Many of the agonists that cause platelet activation are thought to do so by interacting with G protein-coupled receptors on the platelet surface. By activating heterotrimeric G proteins, these receptors evoke shape change, granule secretion and platelet aggregation. This review provides a brief overview of these events, summarizes current information about the role of pleckstrin in events downstream from G protein-coupled receptors, and briefly considers the signaling pathways that couple G protein activation to the low molecular weight GTP-binding proteins which control cytoskeletal reorganization and fibrinogen receptor exposure during platelet activation.

Inhibition of ADP-induced Platelet Responses by Covalent Modification of Aggregin, a Putative ADP Receptor, by 8-(4-Bromo-2,3-dioxobutylthio)ADP

ADP is an important platelet agonist which initiates platelet shape change, aggregation, exposure of fibrinogen receptors, and calcium mobilization. Because of the limitations of previously used affinity analogs and photo-labeling studies as well as controversies surrounding the identity of an ADP receptor on platelets, we have used an affinity label capable of alkylating a putative exofacial receptor on platelets. We now report that 8-(4-bromo-2,3-dioxobutylthio)adenosine-5'-diphosphate (8-BDB-TADP), which is an analog of the natural ligand ADP, blocked ADP-induced platelet shape change, aggregation, exposure of fibrinogen-binding sites, secretion, and calcium mobilization. Following modification by 8-BDB-TADP, the rates of aggregation of platelets induced by thrombin, a calcium ionophore (A23187) or a stimulator of protein kinase C (phorbol myristate acetate) were minimally affected. However, the 8-BDB-TADP-modified platelets exhibited decreased rates of aggregation in response to ADP, as well as collagen and a thromboxane mimetic (U46619), both of which partially require ADP. Autoradiograms of the gels obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized platelets modified by either [beta-32P]8-BDB-TADP, or 8-BDB-TADP and NaB[3H]4 showed the presence of a single radiolabeled protein band at 100 kDa. The intensity of this band was reduced when platelets were preincubated with ADP, ATP, and 8-bromo-ADP prior to labeling by the radioactive 8-BDB-TADP. The results show that 8-BDB-TADP selectively and covalently labeled aggregin (100 kDa), a putative ADP receptor, resulting in a loss of ADP-induced platelet responses.

Protein kinase C activation is not a key step in ADP-mediated exposure of fibrinogen receptors on human platelets

A selective inhibitor of protein kinase C (PKC), Ro 31-8220, blocks pleckstrin (P47) phosphorylation in platelets activated with either ADP, ADP plus synthetic thromboxane agonist U46619 and ADP plus U46619 plus epinephrine, while inducing a weak inhibition of platelet aggregation, and no significant effect on the fibrinogen binding. In platelets activated by U46619 alone, P47 phosphorylation, platelet aggregation, fibrinogen binding and serotonin release are all inhibited by Ro 318220. In the presence of an ADP scavenger system, U46619 induces pleckstrin phosphorylation, serotonin release and calcium mobilization but not platelet aggregation and fibrinogen binding, unless epinephrine is added. In conclusion: (1) PKC activation is required for ADP secretion; (2) ADP or epinephrine are essential for fibrinogen receptor exposure induced by U46619; (3) fibrinogen receptor exposure induced by ADP is independent of activation of PKC.

Calyculin A Inhibits the Exposure of Fibrinogen Receptor in Thrombin-Stimulated Platelets

Calyculin A (CLA) and okadaic acid (OA), specific and potent inhibitors of protein phosphatase 1/2A, inhibit platelet aggregation. However, their inhibitory mechanisms remain unknown. We investigated the effects of CLA on the exposure of fibrinogen receptor in thrombin-stimulated platelets, using flow cytometry with a monoclonal antibody against the fibrinogen receptor of activated glycoprotein (Gp)IIb/IIIa complex (PAC-1). CLA inhibited the exposure of fibrinogen receptor in a dose related manner when added either before or 3 min after thrombin stimulation. In contrast, CLA had no significant effect when the expression of GpIIb/IIIa complex was examined in resting platelets, using a monoclonal antibody recognizing non-activated GpIIb/IIIa complex (NNKY1-32). These results suggest that protein phosphatase 1/2A may be directly involved in the exposure of platelet fibrinogen receptor.

Exposure of Platelet Fibrinogen Receptors by Zinc Ions: Role of Protein Kinase C

Previous studies demonstrated that Zn2+ at a concentration of 50 microM increases the number of fibrinogen receptors exposed on ADP-stimulated platelets and that higher concentrations of Zn2+ induce platelet aggregation that appears to be mediated by receptors associated with the glycoprotein IIb/IIIa complex. The purpose of this study was to identify the mechanism by which Zn2+ modulates exposure of fibrinogen receptors on the surface of human washed platelets. We determined that Zn2+ (300-800 microM)-induced platelet aggregation that was not accompanied by the release of [14C]serotonin was not blocked by ADP scavenging enzymes and 5'-p-fluorosulfonylbenzoyl-adenosine, an affinity label for ADP binding sites, but it was inhibited by disintegrins, staurosporine, and EDTA. Zn2+ (50-200 microM) showed a synergistic effect on platelet aggregation and platelet release caused by ADP and N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine, a Zn2+ chelator, and inhibited ADP-induced platelet aggregation that was reversed by Zn2+ (50 microM). Zn2+ (200 microM) increased the number of fibrinogen binding sites and the affinity of albolabrin (a disintegrin isolated from Trimeresurus albolabris snake venom that has been shown to bind to the fibrinogen receptor) on ADP-activated platelets. On the other hand, Zn2+ (100-800 microM) did not increase fibrinogen binding to the purified receptor. Incubation of platelets with Zn2+ (200 microM) resulted in the phosphorylation of a 47-kDa protein that was blocked by staurosporine, an inhibitor of protein kinase C. In conclusion, Zn2+ ions activate protein kinase C and enhance fibrinogen receptor exposure on the surface of platelets stimulated by ADP.

Evidence for direct coupling of primary agonist-receptor interaction to the exposure of functional IIb–IIIa complexes in human blood platelets. Results from studies with the antiplatelet compound ajoene

Ajoene, ( E, Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide, is a potent antiplatelet compound isolated from alcoholic extracts of garlic. In vitro, ajoene reversibly inhibits platelet aggregation as well as the release reaction induced by all known agonists. In this paper we show that ajoene has a unique locus of action, that is not shared by any other known antiplatelet compound. For example, ajoene inhibits agonist-induced exposure of fibrinogen receptors, as well as intracellular responses such as activation of protein kinase C and the increase in cytoplasmic free calcium induced by receptor-dependent agonists (collagen, ADP, PAF, low-dose thrombin). On the other hand, with agonists that can by-pass (at least partially) the receptor-transductor-effector sequence, such as high-dose thrombin, PMA, NaF, only the exposure of fibrinogen receptors is blocked by ajoene. Binding of fibrinogen to chymotrypsin-treated platelets is only slightly inhibited by ajoene. The results reported here also show that: (a) ajoene does not act as a calcium chelator, does not impair the initial agonist-receptor interaction and does not influence the basal levels of intracellular inhibitors of platelet activation such as cyclic GMP; (b) the locus of action of ajoene is a yet unknown molecular step that links, in the case of physiological agonists, specific agonist-receptor complexes to the sequence of the signal transduction system on the plasma membrane of platelets. In the case of non-physiological, receptor-independent agonists (PMA, NaF), we can only speculate on the hypothesis that they somehow mimic the effect of the agonist-receptor complexes on the signal transduction system; and (c) the exposure of fibrinogen receptors is not a direct consequence of other intracellular processes. These observations clearly show, for the first time, that the exposure of fibrinogen receptors is a membrane event proximally and obligatorily coupled to the occupancy of other membrane receptors by their agonists without any intervention by the cytoplasmic biochemical processes. Additional results support the involvement of G-proteins in these early events of platelet activation. Furthermore, a role of the βτ subunits of G-proteins in the exposure of fibrinogen receptors is proposed.

Activation-independent exposure of the GPIIb-IIIa fibrinogen receptor

Following platelet activation, surface receptors for fibrinogen are exposed. On the activated platelet, glycoprotein IIb-IIIa (GPIIb-IIIa) serves as the receptor for fibrinogen. However, the molecular mechanisms which regulate GPIIb-IIIa fibrinogen receptor exposure are unknown. D3GP3 is an IgG 1, κ monoclonal antibody which is specific for glycoprotein IIIa (GPIIIa). The binding of D3GP3 to GPIIIa, in intact GPIIb-IIIa complexes, induces fibrinogen binding and platelet aggregation. To determine if D3GP3 binding to GPIIIa directly caused the exposure of fibrinogen receptors or, secondarily, due to stimulus response coupling, platelet activation parameters were monitored following the addition of D3GP3 to platelets suspensions. D3GP3 binding did not induce detectable Ca ++ mobilization, protein phosphorylation or activation of the pertussis toxin sensitive G-protein subunit α-41. Further, D3GP3-induced aggregation was not blocked by PGE 1, aspirin, apyrase or the combination of all three reagents. Scanning electron microscopy of D3GP3-induced aggregates demonstrated that the aggregates were composed of discoid platelets. These data suggest that the binding of D3GP3 to GPIIIa induced a conformational change in GPIIb-IIIa such that the fibrinogen receptor was exposed in an activation-independent fashion. This provides evidence that conformational changes in the GPIIb-IIIa complex can result in the transformation of the complex to the high affinity binding competent state.

Cyclosporin A augments human platelet sensitivity to aggregating agents by increasing fibrinogen receptor availability

Clinical use of cyclosporin A (CsA) has been associated with platelet hypersensitivity and an increased incidence of thrombotic and vasoactive events. The purpose of this study was (1) to confirm that CsA enhances platelet sensitivity to the soluble agonists, adenosine diphosphate (ADP) and epinephrine (EPI), and (2) to determine if this enhancement is mediated by alteration in the availability of platelet surface fibrinogen receptors, a final mediator of platelet activation. Mean log dose of ADP required to achieve complete second-wave platelet aggregation in vitro decreased from 1.90 to 1.49 μ M ( n = 19, paired t test, P < 0.05) and 2.86 to 2.11 μ M ( n = 16, P < 0.05) following a 15-min and 3-hr incubation in the absence (saline) and presence of CsA (1000 ng/ml), respectively. At the threshold dose of ADP, concurrent thromboxane B 2 levels at 15 min were 245 ± 44 ng/ml ( n = 12, saline) and 265 ± 54 ng/ml ( n = 9, CsA; P > 0.05). At 3 hr respective levels were 333 ± 57 and 442 ± 81 ng/ml ( P > 0.05). Similar results were obtained with EPI. The number of fibrinogen binding sites in response to 50 μ M ADP was determined in washed platelets in the absence and presence of CsA by radioligand binding. In 6 of 7 volunteers, CsA increased fibrinogen receptors from 26,635 ± 4841 to 35,925 ± 7290 sites/platelet ( x ± SEM; P < 0.05 ). No change in receptor affinity was noted. In conclusion, cyclosporine does augment platelet reactivity. Furthermore, this increase in reactivity is mediated by increased exposure of surface fibrinogen receptors. Delineation of this mechanism may lead to a reduction in cyclosporine-induced complications.

Measurement of fibrinogen binding to platelets in whole blood by flow cytometry: a micromethod for the detection of platelet activation

Platelet fibrinogen binding in whole blood has been measured in vitro by flow cytometry using a commercially available, fluorescein isothiocyanate (FITC)-conjugated polyclonal antifibrinogen antibody. Fibrinogen-antifibrinogen immune complexes were formed in experimental conditions approaching antigen-antibody equivalence, but optimal reaction conditions in which their formation was prevented or minimized could be achieved. Immune complex formation was associated with fibrinogen binding to unstimulated platelets but did not significantly affect ADP-induced fibrinogen binding. Half-maximal fibrinogen binding occurred at about 0.4 microM ADP, and ADP-induced fibrinogen binding continued progressively during 20 min incubation with 10 microM ADP. Fibrinogen binding correlated closely with platelet glycoprotein IIb-IIIa expression in members of a family with Glanzmann's thrombasthenia, and, in double labelling experiments, with the binding of PAC1, a monoclonal antibody that binds to GP IIb-IIIa only after the exposure of fibrinogen receptors. These studies show that platelet fibrinogen binding can be reliably measured in whole blood by means of a polyclonal antifibrinogen antibody which does not discriminate between plasma and platelet-bound fibrinogen, despite the presence of an approximately 100-fold excess of the former.