Platelet GPIIb-IIIa Research Articles

Platelet degranulation and glycoprotein IIbIIIa opening are not related to bleeding phenotype in severe haemophilia A patients

Recently we reported data suggesting that platelets could compensate for the bleeding phenotype in severe haemophilia A (HA). The aim of this study was to confirm these results in a larger population with a detailed characterisation of clinical phenotype. Patients with diagnostic severe HA (FVIII:C <1%) were scored for clinical phenotype by integrating data on age at first joint bleed, joint damage, bleeding frequency and FVIII consumption. Phenotype was defined as onset of joint bleeding-score + arthropathy-score + joint bleeding-score + (2* treatment intensity-score). After a washout period of three days, blood was collected for measurement of basal level of platelet activation, platelet reactivity, endothelial cell activation and presence of procoagulant phospholipids in plasma. Thirty-three patients with severe HA were included, 13 patients with a mild, 12 patients with an average and eight patients with a severe clinical phenotype. No relevant differences in basal level of platelet activation, platelet reactivity, endothelial cell activation and procoagulant phospholipids between all three groups were observed. The mean annual FVIII consumption per kg did not correlate with the platelet P-selectin expression and glycoprotein (GP)IIbIIIa activation on platelets. In conclusion, variability in clinical phenotype in patients with diagnostic severe HA is not related to platelet activation or reactivity, measured as platelet degranulation and platelet GPIIbIIIa opening.

Platelet-Mediated Mechanical Tensile Force Influences ADAMTS13 Localization and Regulation Of Thrombus Development At The Site Of Platelet Accumulation

Background The multimeric glycoprotein von Willebrand factor (VWF) mediates platelet adhesion and aggregation at the site of vessel injury. The adhesive property of VWF is regulated by its multimer length, such that ultra large VWF (ULVWF) multimers, newly released from the endothelium, have greater hemostatic activity. multimer size is regulated by the metalloprotease ADAMTS13, which cleaves the A2 domain to reduce VWF multimer size and functional activity. static conditions, VWF maintains a globular conformation and the ADAMTS13 cleavage site is inaccessible. However, the exposure of endothelial-anchored VWF to tensile forces mediated by platelets and hydrodynamic shear enhance the cleavage of VWF by ADAMTS13. releases VWF of optimal hemostatic length from the endothelium into the plasma. We have previously reported using a flow chamber model which demonstrates that in addition to regulating VWF length and activity at the site of release, ADAMTS13 also associates with VWF at the site of thrombus formation. observed that under conditions of high and very high shear, ADAMTS13 reduced the size of thrombus volume., multi-coloured immunostaining revealed that ADAMTS13 co-localized with VWF and platelets at the top and middle layers of the thrombus, in the presence of very high shear. Aim To better understand the mechanism by which ADAMTS13 regulates thrombus size in our flow chamber model, we assessed the contribution of platelet tensile force to the localization of ADAMTS13 at the site of the thrombus. this model, the contributions of platelet GPIb, GPIIbIIIa, and P-selectin to ADAMTS13 localization were observed. Method Full length mouse VWF and ADAMTS13 cDNA were cloned into pCIneo and pcDNA3.1 plasmid, respectively. The gain of platelet GPIb binding mutation V1316M, and loss of GPIIbIIIa binding mutation (RGD to RGG) were introduced by site-directed-mutagenesis. mCherry was cloned at the C terminus of ADAMTS13 with a 12AA linker. Recombinant mVWF and mADAMTS13-mCherry proteins were produced via HEK293T cells by calcium phosphate transient transfection. mADAMTS13-mCherry (2 U/mL) and wild type or mutant mVWF (4 U/mL) was added to whole blood obtained from VWF-/-/ADAMTS13-/- double knockout mice. Whole blood containing DiOC6-labeled platelets was perfused over a collagen coated flow chamber at very high shear (7500s-1). The role of P-selectin was also analyzed by adding a P-selectin blocking antibody to blood obtained from ADAMTS13-/-knockout mice prior to the flow chamber experiment. After the perfusion, thrombi were fixed and immunostaining was performed to further analyze the distribution of platelets, VWF and ADAMTS13. Result As previously reported, ADAMTS13 localization was observed in the top and middle layers of the thrombus in the presence of wild type mVWF. The GPIb gain-of-function mutation V1316M increased both platelet (126%, p&lt;0.0001) and VWF (190% and p&lt;0.0001) accumulation at the thrombus site. ADAMTS13 localization was also increased (135%, p&lt;0.001) relative to the binding to wild type VWF. Interestingly, with this gain-of-function VWF mutant, ADAMTS13 localization was found throughout the entire thrombus. In contrast, the GPIIbIIIa RGD binding mutant demonstrated decreased VWF (56%, p&lt;0.01), and ADAMTS13 (82%, p&lt;0.05) intensity, although platelet intensity was unaffected. to wild type, ADAMTS13 localized to the middle and top layers of the thrombus. Finally, inhibition of P-selectin significantly decreased VWF (46%, p&lt;0.01) and ADAMTS13 (34%, p&lt;0.01) localization to the thrombus, but again did not significantly alter platelet binding. Conclusion These studies demonstrate the central role of platelet-mediated mechanical tensile force on the regulation of thrombus growth at the site of platelet accumulation. Enhanced tensile force induced by increased GPIb binding resulted in increased ADAMTS13 localization, while reduced tensile force through loss of GPIIbIIIa or P-selectin binding decreased ADAMTS13 localization. This suggests that ADAMTS13 activity at the site of thrombus formation is maintained by the combination of hydrodynamic shear force and platelet tethering. aggregate, these studies suggest that under conditions of shear, ADAMTS13 regulates thrombus size by preserving the hemostatic function of the thrombus, and preventing dysregulated thrombus growth. Disclosures: No relevant conflicts of interest to declare.

Possible Role Of Circulating Adenine Nucleotides and Platelet Activation In The Pathogenesis Of Sickle Cell Disease: Observations In a Murine Model

Vaso-occlusive crisis (VOC) is common in patients with sickle cell disease (SCD). Although hemolysis of erythrocytes has been associated with VOC, the underlying mechanism remains unclear. In the present study, we conducted pathophysiological analyses in Berkeley SCD mice to examine the possible role of adenine nucleotides and platelet activation in SCD. Berkeley SCD mice are genetically modified mice whose murine α-globin and β-globin genes have been deleted, and replaced with human α-, γ-, and δ-globins and sickle β-globin. Control mice were heterozygous littermates expressing normal β-globin.Histological analyses revealed congestion of blood vessels in the liver vasculature and renal medulla of 7-week-old SCD mice associated with aggregated sickle-shaped erythrocytes. Further analysis revealed severe hemolytic anemia in these 7-week-old SCD mice. Plasma adenine nucleotide measurements using LC-MS/MS showed that the concentrations of ADP in 7-week-old SCD mice were significantly higher (∼2.7-fold increase) compared with age-matched control mice. These findings suggest that the intravascular hemolysis of sickled erythrocytes and/or congestion in various organs results in the elevation of circulating concentrations of ADP in this SCD model.Platelet function was assessed by flow-cytometric analysis of platelet-surface activated GPIIbIIIa and P-selectin. These biomarkers of platelet activation were determined in vehicle-treated control mice, vehicle-treated SCD mice, and prasugrel (1 mg/kg/day)-treated SCD mice. Vehicle or prasugrel were orally administered to 7-week-old mice for 1 week and 8 weeks. The expression of activated GPIIbIIIa on platelets was assessed with or without 20 μM ADP stimulation. The expression of platelet P-selectin was assessed with or without 300 μM PAR4 TRAP. These platelet function analyses showed that circulating platelets in SCD mice were activated compared to control mice; the basal expression of activated GPIIbIIIa on platelets in 8- and 15-week-old SCD mice and P-selectin on platelets in 15-week-old SCD mice were increased significantly compared with those in control mice. In vitro stimulation with ADP and PAR4 TRAP increased the expression of activated GPIIbIIIa and P-selectin on platelets in SCD mice. Of note, agonist-stimulated activation was enhanced with age. In 8-week-old mice, the extent of agonist-induced activation of GPIIbIIIa and P-selectin in SCD mice was equivalent to that in control mice; however, in 15-week-old mice, the extent of agonist-induced activation of platelet GPIIbIIIa and P-selectin in SCD mice was significantly higher compared with that in age matched control mice. Prasugrel treatment for 1 week and 8 weeks did not result in significant inhibition of basal expression of activated GPIIbIIIa or P-selectin on circulating platelets in SCD mice, suggesting P2Y12 independent activation of circulating platelets. In contrast, prasugrel treatment for 1 week and 8 weeks did result in highly significant inhibition of ADP-induced GPIIbIIIa activation on SCD mouse platelets. PAR4 TRAP-induced P-selectin expression on SCD mouse platelets was also inhibited by prasugrel.In conclusion, in a murine model of SCD, the present study found significant elevation of circulating ADP possibly resulting from the in vivo hemolysis of sickled erythrocytes. However, P2Y12 inhibition by prasugrel did not appear to reduce the activation of circulating platelets in this SCD model. In contrast, ex vivo stimulation of SCD mouse platelets by the ex vivo addition of ADP and PAR4 TRAP was effectively inhibited by prasugrel administration. The ex vivo addition of ADP may mimic increased exposure to ADP in vivo during VOC thus suggesting a role for prasugrel in the treatment of SCD. Disclosures:Ohno:Daiichi Sankyo Co., Ltd.: Employment. Tanaka:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Samata:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Atsuyuki:Daiichi Sankyo Co., Ltd.: Employment. Mizuno:Daiichi Sankyo Co., Ltd.: Employment. Jakubowski:Eli Lilly and Company: Employment. Sugidachi:Daiichi Sankyo Co., Ltd.: Employment.

Novel Murine Models Of Fetal and Neonatal Alloimmune Thrombocytopenia Established In αIIb Deficient and Human αIIb Transgenic Mice

BackgroundFetal and neonatal alloimmune thrombocytopenia (FNAIT) is a severe bleeding disorder that often results in intracranial hemorrhage (ICH), leading to neurological impairments or death. FNAIT has also been linked with miscarriage although the incidence of fetal loss has not been adequately studied. It is the most common cause of severe thrombocytopenia in live born neonates and accounts for up to 35% of all neonates admitted into the intensive care unit. In FNAIT, maternal alloantibodies cross the placenta and target paternally-derived fetal platelet antigens, most commonly platelet GPIIbIIIa (αIIbβ3 integrin). Polymorphisms in human platelet antigen-1a (HPA-1a), located on the integrin β3 subunit are the most common cause of FNAIT and are most widely studied, however the reported incidence of anti-αIIb-mediated FNAIT has recently increased; but the pathogenesis of this disease is unclear. Methods and ResultsTo establish mouse models of anti-αIIb antibody mediated FNAIT, αIIb deficient (αIIb-/-) and human αIIb transgenic mice were employed in this study. We first immunized female αIIb-/- mice by weekly transfusions of wild type (WT) mouse platelets 2 times or 4 times. We demonstrated that the αIIb-/- mice were immunoresponsive to the αIIb antigen and both IgG1 and IgG2 antibodies against αIIb (i.e.TH2-like and TH1-like immune response, respectively) were detected two weeks after platelet transfusion. To test whether αIIb antisera can cause platelet destruction, we injected the antisera into WT mice and found severe thrombocytopenia in the recipient mice (P<0.001). Subsequently, immunized female αIIb-/- mice were bred with WT males to generate heterozygous fetuses and to recapitulate the features of FNAIT. Fetuses and neonates generated from naïve αIIb-/- females bred with WT males were used as a control.We found the severity of symptoms was correlated with the level of antibody titers in the maternal circulation. In 2 times and 4 times immunized females, miscarriage was observed (n= 2/9; and n =3/3, respectively). Upon post mortem dissection of miscarrying mothers at embryonic day 15.5, fetuses possessed ICH in 2 times and 4 times immunized females (n=2/14; and 5/12, respectively). Live born neonates from 2 times immunized mothers showed severe thrombocytopenia (P<0.001), purpura (n=9/22) and ICH (n=4/22). In addition, neonate body weight decreased compared to naïve controls (P<0.05), suggesting intrauterine growth restriction. There were no live pups delivered from 4 times immunized mothers. We also tested the effect of antisera from 2 times immunized αIIb-/- mice on platelet function and found the antisera enhanced ADP-induced platelet aggregation. This is different from the anti-β3 sera that inhibited platelet aggregation in our previous FNAIT model. Whether the different effects on platelets may lead to different pathology in FNAIT is under investigation in the laboratory.In addition to the knockout mouse model, we also used human αIIb transgenic mice, which do not express mouse αIIb on their platelets, in order to generate an alloimmune model of FNAIT. This model may better mimic the conditions of human FNAIT caused by the polymorphisms in αIIb genes. Following the same protocol as we used in the αIIb-/- mice, we found anti-murine αIIb antibodies were induced although the antibody titer was slightly lower. After breeding with male WT mice, immunized female human αIIb transgenic mice deliver pups with thrombocytopenia and ICH. Miscarriage was also observed, however the severity and symptoms of the disease were less. Furthermore, we also established another model of mouse-anti-human FNAIT by breeding humanized αIIb male mice with immunized female WT mice. We will compare the differences in these iso- and alloimmune models to elucidate the pathogenesis of αIIb mediated FNAIT. Conclusions and SignificanceWe established the first anti-αIIb model of FNAIT and the first alloimmune animal models of FNAIT. Our results indicate that these models recapitulate human disease symptoms such as; lower platelet counts, bleeding diasthesis, and decreased birth weight compared to controls. Both neonates and fetuses possess ICH, and some immunized mothers underwent miscarriage. These models give us the opportunity to compare iso- vs. alloimmune FNAIT, and to compare the pathology between anti- αIIb and our anti-β3 mediated FNAIT. Disclosures:No relevant conflicts of interest to declare.

TCT-142 Prasugrel 5 mg inhibits platelet GPIIb-IIIa and P-Selectin expression in the very elderly - Results from the GENERATIONS trial, a pharmacodynamic study in stable CAD patients

Platelet surface P-selectin and activated GPIIb-IIIa are markers of platelet activation, degranulation and aggregation. In the TRITON trial prasugrel (Pras) 10 mg reduced ischemic events vs. clopidogrel (Clop) 75 mg but increased bleeding, notably in very elderly (VE) patients. Pras 5 mg is a

An ω-6 Fatty Acid, Dgla, Prevents Platelet Activation and Thrombosis in Vivo.

AbstractAbstract 2169Platelets play a pivotal role in thrombotic events leading to clot formation and clot stability in vivo. Uncontrolled signaling events in the platelet can result in unwanted thrombosis, which may eventually lead to the development of myocardial infarction or stroke. Previously, we have shown that ex vivo treatment of human platelets with the ω-6 fatty acid dihomo-γ-linolenic acid (DGLA) or its eicosanoid derived from 12-LOX oxidation, 12-hydroxyeicosatrienoic acid (12(S)-HETrE), inhibits PAR and collagen-induced platelet aggregation, clot retraction, and GPIIbIIIa activation. Since early studies have shown dietary supplementation of fatty acids increase fatty acid incorporation into the platelet lipid membrane (Barre, DE Lipids 1992; 27(5): 315–320; Marry, MJ Prostaglandins Leukot Essent Fatty Acids 1997; 56(3):223–223), we postulated that altering fatty acid composition in the platelet through dietary supplementation in vivo may be a viable approach to inhibiting platelet function. Therefore, a longitudinal study of wild-type mice on normal chow compared to mice supplemented with high (.5 g/kg) or low (.13 g/kg) DGLA diets was conducted. Each set of mice (7–8 mice) was given the designated diet for a period of 1, 2, or 3 months. At each time point, tail bleeding times and ex vivo platelet function in PRP were performed. Tail bleeding times from mice on the high DGLA diet were significantly prolonged by more than 15 minutes. Further, a smaller but statistically significant delay in clotting time was observed in mice on the low DGLA diet compared with control mice. Additionally, ex vivo aggregation response to collagen (1 μg/mL to 20 μg/mL) and PAR4-AP (50 μM to 500 μM) in platelets from mice on the high DGLA diet showed significant shifts to the right in their ability to induce platelet aggregation compared with control mice suggesting these mice were protected against thrombosis. JON/A and P-selectin binding to the PRP of high and low DGLA were also significantly attenuated in response to PAR4-AP. This study, which evaluated the in vivo and ex vivo effects of DGLA on regulation of platelet reactivity, supports DGLA as a potent, endogenous anti-thrombotic agent. Understanding the mechanistic details by which DGLA protects against thrombosis and maintains hemostasis through its COX-1 and 12-LOX-dependent bioactive metabolites will help to identify the potential viability of this target for anti-platelet intervention. Disclosures:No relevant conflicts of interest to declare.

Mechanisms of C-reactive protein, interleukin-6 and soluble CD40L blood concentration changes after coronary stent implantation

Background: The pathway linking inflammation and thrombosis has been extensively studied. Experimental data support that arterial thrombosis also induces a detectable inflammatory response, which in turn, activates prothrombotic pathways closing a vicious circle that interconnects inflammation and thrombosis. Aim: We designed this study to investigate the causes of inflammatory markers increase after coronary angioplasty. Methods: We analyzed the interrelationship of thrombotic and inflammatory markers and the effect of blocking thrombus formation on the inflammatory response in 50 patients undergoing high thrombotic risk coronary angioplasty. The relationship of platelet number to soluble CD40 Ligand, Interleukin-6 and C-reactive protein blood levels was studied. Half of the study population was treated with standard antithrombotic drugs and the other half with the standard therapy plus platelet GP IIb-IIIa receptor inhibitor Eptifibatide. Results: There was a clear correlation between basal platelet count and sCD40L basal levels, post-angioplasty sCD40L increase and post-angioplasty IL-6 levels and post-angioplasty IL-6 levels with post-angioplasty CRP levels. Postangioplasty CRP, IL-6 and sCD40L blood levels were influenced by GP IIb-IIIa treatment in patients with angiographic thrombus. Conclusion: Platelet aggregation induces a proinflammatory response which is blocked by a GP IIb-IIIa inhibitor agent, particularly in patients with patent angiographic thrombus.

Relative efficacy of steroid therapy in immune thrombocytopenia mediated by anti‐platelet GPIIbIIIa versus GPIbα antibodies

Immune thrombocytopenia (ITP) is characterized by platelet clearance mediated primarily by autoantibodies against the platelet GPIIbIIIa and/or GPIbα. Steroid therapy is a first-line treatment for ITP. However, some patients are refractory to this therapy and currently no method can predict which patients will respond. To evaluate whether steroids are equally efficacious in treating patients with ITP caused by anti-GPIIbIIIa versus anti-GPIbα antibodies, we performed a retrospective study on 176 newly diagnosed patients with acute ITP who had severe bleeding symptoms and were admitted as resident patients to the hospital. The patients were treated first with intravenous administration of high-dose dexamethasone (DXM), followed by oral administration of prednisone. Response to therapy was observed in a majority of patients with antibodies specific for GPIIbIIIa (31/43) or without detectable antibodies against either GPIIbIIIa or GPIbα (36/45). In contrast, the steroid response was significantly lower in patients with anti-GPIbα antibodies (9/34) or with antibodies against both GPIbα and GPIIbIIIa (16/54). The preliminary findings of this study suggest that in future prospective clinical trials including corticosteroids, the anti-GPIbα, and -GPIIbIIIa status should be assessed in order to test its potential relevance in deciding future treatments.

Apoptosis in the anucleate platelet

For many years, programmed cell death, known as apoptosis, was attributed exclusively to nucleated cells. Currently, however, apoptosis is also well-documented in anucleate platelets. This review describes extrinsic and intrinsic pathways of apoptosis in nucleated cells and in platelets, platelet apoptosis induced by multiple chemical stimuli and shear stresses, markers of platelet apoptosis, mitochodrial control of platelet apoptosis, and apoptosis mediated by platelet surface receptors PAR-1, GPIIbIIIa and GPIbα. In addition, this review presents data on platelet apoptosis provoked by aging of platelets in vitro during platelet storage, platelet apoptosis in pathological settings in humans and animal models, and inhibition of platelet apoptosis by cyclosporin A, intravenous immunoglobulin and GPIIbIIIa antagonist drugs.

Poly-N-Acetyl Glucosamine Fibers Accelerate Hemostasis in Patients Treated With Antiplatelet Drugs

Nanofibers consisting of poly-N-acetyl glucosamine (pGlcNAc), as the functional component of products for surface hemostasis, have been shown to activate platelets and thereby the clotting mechanism. The nanofiber-activated platelets provide a catalytic surface for acceleration of the intrinsic coagulation cascade, thrombin generation, and fibrin polymerization. Thromboelastographic analysis was undertaken to study the role of the pGlcNAc nanofibers in platelet activation and acceleration of fibrin polymerization. Thromboelastographic studies were performed without added activators of coagulation. The pGlcNAc nanofibers were found to accelerate fibrin polymerization in whole blood and platelet-rich plasma. Treatment with eptifibatide (an inhibitor of the platelet GPIIbIIIa receptor) and corn trypsin inhibitor inhibited clotting of whole blood and platelet-rich plasma. The inhibition was reversed by treatment with pGlcNAc nanofibers. Inhibition was not observed after treatment with aspirin alone, MRS2359 (platelet ADP receptor inhibitor), or by a combination of aspirin and MRS2359. The pGlcNAc nanofibers accelerate clotting in normal blood treated with aspirin and MRS2359. Clopidogrel (Plavix) and aspirin did not affect the kinetics of pGlcNAc-mediated fibrin polymerization in blood from patients treated with antiplatelet drugs compared with nontreated blood. These results provide evidence that pGlcNAc nanofibers activate platelets and accelerate the clotting of blood, and on how best to achieve surface hemostasis when patients are coagulopathic because of shock and/or to treatment with antiplatelet drugs.

The 9p21.3 Locus

A series of reports in 2007 established that single nucleotide polymorphisms (SNPs) in the 9p21.3 locus are associated with coronary heart disease (CHD), both in patients with acute myocardial infarction and chronic atherosclerosis.1,–,4 Subsequently, many additional studies used a candidate locus approach to replicate the 9p21.3-CHD association. Intriguingly, other phenotypes have also been found to be associated with this genomic region, including type II diabetes, stroke, malignant melanoma, aortic aneurysms, cerebral aneurysms, and periodontitis. Unfortunately, neither the causative variants nor genes have been established. Although genetic associations may permit better disease risk stratification, without the knowledge of the genes and variants we lose opportunities to improve our basic understanding of the disease process and hence the potential for targeted therapies. Article see p 445 Many cellular pathways in multiple tissues contribute to the pathogenic processes resulting in CHD. There is value in using intermediate phenotypes as outcomes in genetic association studies because there is enhanced power to detect gene associations when the number of genes potentially responsible for the phenotype is reduced, thereby increasing the fraction of the variance explained by any single factor or gene. In addition, intermediate traits are usually easier to define (have less heterogeneity) than clinical disease. Somewhat surprisingly, 9p21.3 has not been associated with measures believed to represent the chronic process of atherosclerosis or endothelial cell reactivity,5 suggesting that this locus may contribute to CHD by an alternate pathophysiologic mechanism. There is abundant pathological and clinical evidence demonstrating the vital role played by blood platelets in acute coronary syndromes, which result from the formation of occlusive platelet thrombi in coronary arteries at the sites of ruptured atherosclerotic plaques,6,7 and antiplatelet agents have become a mainstay of therapy in patients with acute coronary syndromes. In addition to …

Intracoronary Shear-Related Upregulation of Platelet P-selectin, Platelet-Monocyte Aggregates and Monocyte CD11b, but not Platelet GPIIb-IIIa Activation in Humans Despite the Use of Aspirin and Clopidogrel

Results: In comparison to 4th generation testing, the “7/10” and “10/10” change criteria showed improved clinical sensitivity and the same or improved clinical specificity for the diagnosis of Myocardial Infarction. The “White” criteria showed decreased clinical sensitivity but better specificity. Criterion used Sensitivity (95% CI) Specificity (95% CI) TnT (4th generation) 92.86 (80.99–97.54) 91.51 (84.65–95.47) “7/10” criteria for hsTnT 95.24 (84.21–98.68) 91.51 (84.65–95.47) “10/10” criteria for hsTnT 95.24 (84.21–98.68) 92.45 (85.81–96.13) “White” criteria for hsTnT 90.48 (77.93–96.23) 95.28 (89.43–97.97) Conclusion: The use of the hsTnT assay has the potential to improve clinical sensitivity and specificity in the diagnosis of myocardial infarction. doi:10.1016/j.hlc.2010.06.731

Neutrophil Platelet Satellitism Revisited: Sidedness and Domain of Neutrophil Associated Platelet Aggregates.

Abstract 4469Platelet Satellitism surrounding polymorphonuclear neutrophils has been observed almost exclusively in EDTA-treated blood at room temperature. The mechanism underlying this phenomenon is not fully understood. In a PubMed search of the English language medical literature, there are only 44 reported cases involving the phenomenon of Platelet Satellitism.We report a case of platelet rosetting around neutrophils in a 78-year old woman with incidental thrombocytopenia. Her isolated thrombocytopenia was not mediated by any form of immunosuppression, medications, hypersplenism, intravascular consumption, or diminished platelet production. A diagnosis of Pseudothrombocytopenia was made based upon her peripheral blood smear revealing platelet aggregates displaying sidedness in relation to her neutrophils.Platelet Satellitism is postulated to represent an immunologic phenomenon caused by the presence of natural antibodies in which the platelets aggregate around polymorphonuclear neutrophils. The reasons behind why certain individuals possess agglutinating antibodies that lead to platelet clumping, and others have antibodies that cause platelet satellitism is unknown. A proposed mechanism is natural antibodies directed against different epitopes on the platelet GPIIb-IIIa complex. The reported frequency of platelet Satellitism is much lower than that of EDTA platelet clumping (approximately 1:30,000 blood counts) according to the reviewed literature.Platelet Satellitism to polymorphonuclear neutrophils was initially documented by Field and MacLeod in 1963, and has since been reported as an incidental finding in peripheral blood smears when EDTA was used as an anticoagulant. The process by which platelets bind and form rosettes around polymorphonuclear leukocytes is due to activation of EDTA-dependent antiplatelet and antineutrophil IgG autoantibodies directed against the platelet glycoprotein IIb/IIIa complex and Fc receptors of neutrophils. Further, it is theorized that a non-immunologic cause may play in role in which adherence is induced by thrombospondin or the alpha-granule protein of other platelets. In rare instances, platelets may aggregate around monocytes or basophils.Our retrospective review underscores the importance of recognizing the principle of Pseudothrombocytopenia due to EDTA-induced Platelet Satellitism. This entity is in vitro phenomena which has no clinical bearing in terms of a predisposition to increased mucous membrane bleeding. As in other literature cases, a clear correlation between the presence of IgG antibodies and a specific clinical situation, disease, or use of drugs could not be demonstrated. Therefore, these antibodies, which are present in some normal individuals, might occur naturally. Due to the exposure of certain antigenic structures present on EDTA-modified platelets and neutrophils, they may manifest themselves by triggering the Platelet Satellitism phenomenon. Disclosures:No relevant conflicts of interest to declare.

Arterial thrombosis: relevance of a model with two levels of severity assessed by histologic, ultrastructural and functional characterization.

We previously described a model of laser-induced thrombosis in mesenteric arterioles with superficial and deep levels of injury producing a transient thrombus resolving within 2 min and a larger almost occlusive thrombus, respectively. Both types of lesion were sensitive to platelet GPIIb-IIIa and P2Y(12) inhibition, whereas only deep injuries were sensitive to thrombin blockade. The aim of the present study was to use histologic methods and electron and intravital microscopy to characterize the lesions and thrombi and to extend our knowledge of the sensitivity of this model to genetic and pharmacologic inhibition. A superficial injury was found to detach the endothelial cells and expose a collagen III- and IV-rich subendothelium where platelets could adhere. Tissue factor and fibrin were not detected. Deeper penetration of the external elastic lamina occurred in deep injuries, with exposure of collagen I, III and IV. Here the thrombus was composed of platelets exhibiting a decreasing gradient of degranulation from the deepest lesion area to the surface. Fibrin was found close to the most activated platelets. Consistently, glycoprotein VI (GPVI)-collagen and GPIb-von Willebrand factor (VWF) interactions were found to be critical in superficial injuries. After deep lesion, thrombus formation was modestly reduced in GPVI-immunodepleted mice and still strongly inhibited in VWF(-/-) mice. Combined hirudin infusion and GPVI depletion further inhibited thrombosis after deep injury. This study confirms the feasibility of inducing arterial thrombosis with distinct levels of severity and establishes the central roles of collagen and VWF in thrombus formation after superficial injury. Collagen, VWF and thrombin all appear to contribute to thrombosis after deep arterial lesion.

Clinically Relevant Concentrations of Dopamine Do Not Amplify Agonist-induced Human Platelet Ca2+ Mobilization or GP IIb IIIa Activation and Do Not Accelerate Acute Coronary Thrombosis in Dogs

Dopamine is an inotrope effective in the short term treatment of acute heart failure - including that caused by coronary artery disease. Catecholamines however can potentiate platelet activation and pre-dispose to coronary thrombosis. Dopamine was studied for effect on agonist induced human platelet Ca mobilization, human platelet GP iib iiia receptor activation and acute coronary thrombosis in dogs. Calcium sensitive indo-1, fluorescent immunostaining and flow cytometry were used for platelet studies while coronary thrombosis was induced in anesthetized dogs via endothelial damage, arterial wall injury and critical stenosis. Dopamine 10 and 10 M had no effect on the amplitude of the platelet Ca signal evoked by thrombin 0.1 U/mL. Likewise, dopamine 10 M had no effect on GP IIb IIIa activation evoked by ADP 10 M and by thrombin 0.1 U/mL. In dogs, intravenous dopamine 8 ug/Kg/min had no effect on repetitive cycles of acute coronary thrombus formation. In positive control studies, epinephrine increased platelet responsiveness and accelerated canine coronary thrombosis. Clinically relevant concentrations of dopamine did not amplify agonist induced human platelet Ca activation, GPiib iiia expression or experimental canine coronary thrombosis--providing a degree of reassurance concerning this versatile inotrope.

Drug-induced thrombocytopenia: pathogenesis, evaluation, and management

Although drugs are a common cause of acute immune-mediated thrombocytopenia in adults, the drug etiology is often initially unrecognized. Most cases of drug-induced thrombocytopenia (DITP) are caused by drug-dependent antibodies that are specific for the drug structure and bind tightly to platelets by their Fab regions but only in the presence of the drug. A comprehensive database of 1301 published reports describing 317 drugs, available at www.ouhsc.edu/platelets, provides information on the level of evidence for a causal relation to thrombocytopenia. Typically, DITP occurs 1 to 2 weeks after beginning a new drug or suddenly after a single dose when a drug has previously been taken intermittently. However, severe thrombocytopenia can occur immediately after the first administration of antithrombotic agents that block fibrinogen binding to platelet GP IIb-IIIa, such as abciximab, tirofiban, and eptifibatide. Recovery from DITP usually begins within 1 to 2 days of stopping the drug and is typically complete within a week. Drug-dependent antibodies can persist for many years; therefore, it is important that the drug etiology be confirmed and the drug be avoided thereafter.

Inhibition of platelet GPIIb–IIIa and P-selectin expression by aspirin is impaired by stress hyperglycemia

Increased aspirin resistance may contribute to the increase in thrombotic events observed in patients with type 2 diabetes. In this study, we examined if acute exposure to increased plasma glucose impaired the inhibitory effects of aspirin on platelet activation. Whole-blood samples were incubated with 100 (euglycemia), 200, 300, and 600 mg/dl glucose followed by incubation with aspirin [acetylsalicylic acid (ASA)]. Using flow cytometry, GPIIb–IIIa and P-selectin were analyzed in unstimulated and arachidonic acid (AA)-stimulated platelets. In euglycemic blood, AA caused a significant increase in platelet GPIIb–IIIa expression [unstimulated: 59.5±8.2 total fluorescence intensity (TFI), AA stimulated: 319.6±42.7 TFI, P=.002] and P-selectin (4.4±0.7 and 179.5±38.5 TFI, P<.001). In vitro, ASA significantly inhibited both GPIIb–IIIa expression (36.5%) and P-selectin expression (81%; P<.005). However, increased blood glucose (200 mg/dl) significantly impaired the inhibitory effect of ASA (84% for GPIIb–IIIa, P<.005; 48% for P-selectin, P=NS). Increasing glucose to 600 mg/dl completely overwhelmed the inhibitory effect of ASA. A statistically significant interaction between glucose concentration and ASA dose was found ( P<.001 for GPIIb–IIIa and P=.004 for P-selectin). In vitro, concentration-dependent stress hyperglycemia significantly impaired the inhibitory effects of aspirin on human platelet GPIIb–IIIa and P-selectin expression. Under acute hyperglycemic conditions, the effectiveness of ASA to inhibit platelets via the AA-activation pathway may be significantly reduced.

Efficient platelet δ-granule release induced by [Ca2+]i elevation is modulated by GPIIbIIIa

Intracellular Ca2+ elevation generates a cascade of events that leads to platelet activation and degranulation. The GPIIbIIIa-ligand molecular complex plays a central role in several aspects of platelet activation. Taking advantage of the flow cytometric simultaneous analysis of surface GPIIbIIIa expression and intracellular serotonin content, we demonstrate here that the functional inhibition of GPIIbIIIa generates an impairment of delta-granule release even upon maximal intracellular Ca2+ elevation. In healthy subjects, the GPIIbIIIa inhibitor tirofiban impairs platelet delta-granule release. Analogously, Glanzmann thrombasthenia patients show an impairment of delta-granule release that is proportional to their residual expression of platelet GPIIbIIIa. These data show that platelet surface expression of functional GPIIbIIIa is required for a fully efficient secretion of delta-granules and serotonin release. The implications of our findings are discussed in the light of the complex interplay between vesicle release and ligand-receptor triggering during platelet activation.

Clostridium perfringens phospholipase C-induced platelet/leukocyte interactions impede neutrophil diapedesis

Clostridium perfringens gas gangrene is a fulminant necrotizing infection in which inflammatory cells are notably absent from infected tissues but are often massed within adjacent vessels. It has been shown that C. perfringens phospholipase C (PLC) stimulates formation of large intravascular platelet/leukocyte complexes and that PLC-induced activation of platelet gpIIbIIIa plays a major role. In vivo, such aggregates contribute to microvascular thrombosis and ischaemic necrosis of tissue. However, the effects of adherent platelets on neutrophil diapedesis have not been established. The present work investigated (1) the contribution of platelet P-selectin (CD62P) to PLC-induced cellular complex formation and (2) the effects of platelet adhesion on neutrophil diapedesis. The effects of anti-gpIIbIIIa and anti-CD62P strategies on PLC-induced complex formation were measured by flow cytometry and followed by light microscopy. Both platelet gpIIbIIIa and CD62P contributed to the formation of platelet/leukocyte complexes. Specifically, gpIIbIIIa mediated the formation of large platelet/platelet aggregates that were tethered to the leukocyte principally via CD62P. Neutrophil diapedesis, quantified by a transendothelial cell migration assay and visualized by electron microscopy, was significantly reduced (>60%) by the adherence of large platelet aggregates. It was concluded that the absence of a tissue inflammatory response in C. perfringens gas gangrene is due, in part, to impaired neutrophil mobility caused by large aggregates of adherent platelets induced by PLC. Further, an adjunctive immunotherapeutic strategy targeting both gpIIbIIIa and CD62P may improve the tissue inflammatory response, prevent vascular occlusion, maintain tissue viability, and reduce the need for radical amputation in patients with clostridial gas gangrene.

Chirurgie cardiaque effectuée avec une association héparine non fractionnée et inhibiteur des récepteurs GPIIbIIIa plaquettaire (tirofiban) pour suspicion de TIH

In a patient with heparin-induced thrombocytopenia few antithrombotic alternate treatments are proposed for cardiac surgery with or without cardiopulmonary bypass: danaparoid, lepirudine or powerful antiplatelet agent. Recently, the platelet GPIIbIIIa antagonist tirofiban (Aggrastat ®) was tested in humans. We reported two cases of patients operated upon for cardiac surgery with unfractionnated heparin (UFH) and tirofiban. The first patient underwent an off-pump coronary artery bypass graft and the second one a mitral valvular replacement under cardiopulmonary bypass. Tirofiban was associated with UFH and aprotinine. Postoperative bleeding was in the normal range for the two types of surgeries and haemodynamic tolerance was good. These two case reports support the possibility of secure cardiac surgery under efficient platelet inhibition with tirofiban. The management of cardiac surgery with tirofiban without monitoring of platelet aggregation appeared to be more simple than with the other alternate antithrombotic agents.