GPIIb-IIIa Complex Research Articles

Platelet dysfunction after trauma: From mechanisms to targeted treatment.

Platelet dysfunction after trauma: From mechanisms to targeted treatment.

Increase in GP IIb-IIIa and P2Y12 Receptors in Activated Platelets as the Possible Indicator of de novo Protein Synthesis

Although platelets lack nuclei, they are capable of de novo protein synthesis. We speculate that key platelet receptors are involved in the regulation of this process, and the changes in their number indicate the de novo protein synthesis in platelets. The object of our study was native platelets obtained from healthy donors. Using flow cytometry and Western blot, we determined the number of GP IIb-IIIa receptors (fibrinogen receptor) and P2Y12 receptors (ADP receptor) on the surface of platelets upon their activation with ADP and collagen. To verify the approaches and techniques used, we studied IL-1β protein, which was previously shown to be synthesized de novo in activated platelets. GP IIb-IIIa receptor numbers correlate with the number of P2Y12 receptors on the cell surface (R = 0.45, p = 0.03). It was demonstrated that the platelet receptor numbers are higher on the surface of the cells with high functional activity. According to the data obtained by Western blot, upon the cell activation with ADP, the number of GP IIb-IIIa and P2Y12 receptors increases, which may serve as evidence of these proteins being synthesized in the activated platelets. It was observed that the level of P2Y12 and IL-1β was lower in the samples where GP IIb-IIIa receptor was blocked by the selective inhibitor, i.e., the Fab fragment of the antibodies that specifically recognizes the GP IIb-IIIa complex. This suggests the important role of GP IIb-IIIa receptor in the regulation of protein synthesis.

Clinical effect of super-selective intracoronary administration on acute myocardial infarction patients

Objective To investigate the effect of super-selective intracoronary administration on acute myocardial in farction patients. Methods A total of 240 patients with ST-segment elevation myocardial infarction who received emergency percutaneous coronary intervention in our department from March 2012 to January 2014 were selected and divided into the intravenous drug administration group (n=77), the conventional intracoronary drug administration group (n=81) and the super-selective intracoronary drug administration group (n=82). Parameters, including the Thrombolysis in Myocardial Infarction (TIMI) classification, ST segment resolution after operation, peak values of creatine kinase MB (CK-MB) and troponin-I (cTn-I), left ventricular ejection fraction, left ventricular end-diastolic diameter (LVEDD), major adverse cardiovascular events and bleeding events, were compared between the groups. Results There were no significant differences in TIMI flow grade between the three groups (χ2=0.14, P=0.529). The percentage of patients with complete ST segment resolution after operation was higher in the super-selective intracoronary drug administration group than in the intravenous drug administration and conventional intracoronary drug administration groups (74.4% vs. 62.3%, 61.7%, χ2=8.24, P 0.05). Conclusions Super-selective intracoronary drug administration can significantly enhance cardiac function and alleviate angina pectoris in patients with acute myocardial infarction, and should be a recommended method. Key words: Myocardial infarction; Angioplasty, transluminal, percutaneus coronary; Platelet glycoprotein GPⅡb-Ⅲa complex

Platelet Membrane Glycoproteins: A Historical Review

The search for the components of the platelet surface that mediate platelet adhesion and platelet aggregation began for earnest in the late 1960s when electron microscopy demonstrated the presence of a carbohydrate-rich, negatively charged outer coat that was called the "glycocalyx." Progressively, electrophoretic procedures were developed that identified the major membrane glycoproteins (GP) that constitute this layer. Studies on inherited disorders of platelets then permitted the designation of the major effectors of platelet function. This began with the discovery in Paris that platelets of patients with Glanzmann thrombasthenia, an inherited disorder of platelet aggregation, lacked two major GP. Subsequent studies established the role for the GPIIb-IIIa complex (now known as integrin αIIbβ3) in binding fibrinogen and other adhesive proteins on activated platelets and the formation of the protein bridges that join platelets together in the platelet aggregate. This was quickly followed by the observation that platelets of patients with the Bernard-Soulier syndrome, with macrothrombocytopenia and a distinct disorder of platelet adhesion, lacked the carbohydrate-rich, negatively charged, GPIb. It was shown that GPIb, through its interaction with von Willebrand factor, mediated platelet attachment to injured sites in the vessel wall. What follows is a personal reflection on the studies that were performed in the early pioneering days.

Comparison of cytotoxic and anti-platelet activities of polyphenolic extracts from Arnica montana flowers and Juglans regia husks

Polyphenolic compounds of plant origin are well known to be beneficial to human health: they exert protective effects on haemostasis and have a particular influence on blood platelets. However, the anti-platelet properties of polyphenolic compounds observed so far have not been weighed against their potential cytotoxic action against platelets. The aim of this study was to demonstrate that anti-platelet and cytotoxic effects on blood platelets may interfere and therefore, may often lead to confusion when evaluating the properties of plant extracts or other agents towards blood platelets. The anti-platelet and cytotoxic in vitro effects of plant extracts obtained from the husks of walnuts (J. regia) and flowers of arnica (A. montana) on platelet reactivity and viability were examined. Platelet function was assessed using standard methods (flow cytometry: P-selectin expression, activation of GPIIbIIIa complex, vasodilator-stimulated phosphoprotein, VASP index; turbidimetric and impedance aggregometry) and newly set assays (flow cytometric monitoring of platelet cytotoxicity). The results reveal that none of the studied plant extracts demonstrated cytotoxicity towards blood platelets. The phenolic acid-rich extract of A. montana (7.5 and 15 µg/ml) significantly reduced the ADP-induced aggregation in both whole blood and PRP, and decreased the platelet reactivity index (PRI; VASP phosphorylation) in whole blood, while showing excellent antioxidant capacity. The extract of J. regia husks significantly reduced ADP-induced platelet aggregation in whole blood when applied at 7.5 µg/ml, and only slightly decreased the PRI at 15 µg/ml. Both examined extracts suppressed platelet hyper-reactivity, and such influence did not interfere with cytotoxic effects of the extracts. Thus, its high polyphenol content, excellent antioxidant capacity and distinct anti-platelet properties, in combination with its lack of toxicity, make the extract of A. montana flowers a possible candidate as an anti-platelet agent or a compounding diet supplement.

Glycoprotein IIb/IIIa and P2Y12 induction by oligochitosan accelerates platelet aggregation.

Platelet membrane receptor glycoprotein IIb/IIIa (gpiibiiia) is a receptor detected on platelets. Adenosine diphosphate (ADP) activates gpiibiiia and P2Y12, causing platelet aggregation and thrombus stabilization during blood loss. Chitosan biomaterials were found to promote surface induced hemostasis and were capable of activating blood coagulation cascades by enhancing platelet aggregation. Our current findings show that the activation of the gpiibiiia complex and the major ADP receptor P2Y12 is required for platelet aggregation to reach hemostasis following the adherence of various concentrations of chitosan biomaterials [7% N,O-carboxymethylchitosan (NO-CMC) with 0.45 mL collagen, 8% NO-CMC, oligochitosan (O-C), and oligochitosan 53 (O-C 53)]. We studied gpiibiiia and P2Y12 through flow cytometric analysis and western blotting techniques. The highest expression of gpiibiiia was observed with Lyostypt (74.3 ± 7.82%), followed by O-C (65.5 ± 7.17%). Lyostypt and O-C resulted in gpiibiiia expression increases of 29.2% and 13.9%, respectively, compared with blood alone. Western blot analysis revealed that only O-C 53 upregulated the expression of P2Y12 (1.12 ± 0.03-fold) compared with blood alone. Our findings suggest that the regulation of gpiibiiia and P2Y12 levels could be clinically useful to activate platelets to reach hemostasis. Further, we show that the novel oligochitosan is able to induce the increased expression of gpiibiiia and P2Y12, thus accelerating platelet aggregation in vitro.

Research advances in monitoring platelet function in patients with coronary artery disease

Antiplatelet therapy and stent implantation have been the dominant treatment to reduce the mortality of patients with coronary artery disease.Recently,studies have showed that adverse cardiac events still occur in part of patients with coronary artery disease after the antiplatelet treatment with aspirin and/or clopidogrel.Thus,resistance to aspirin and clopidogrel has attracted increasing attention.It will be great benefit to these patients who were identified resistance and made tailoring antiplatelet therapy So far many platelet function tests has been used in clinical to monitor the reaction of the antiplatelet drugs for prevention and treatment of thrombosis in patients with coronary artery disease.These monitoring tests may be chosen based on different antiplatelet drugs including aspirin,clopidogrel and GPⅡb/Ⅲa antagonist. The results of antiplatelet drug resistance may be different due to different platelet function methods,thus the related clinical adverse events needs further verification.(Chin J Lab Med,2013,36:489-493) Key words: Coronary artery disease; Platelet function tests; Aspirin; Ticlopidine; Plateletglycoprotein GPⅡb-Ⅲa complex

Limitations of the MEDLINE Database in Constructing Meta-analyses

Letters1 March 2011Limitations of the MEDLINE Database in Constructing Meta-analysesDavid E. Winchester, MD and Anthony A. Bavry, MD, MPHDavid E. Winchester, MDFrom University of Florida, Gainesville, FL 32610.Search for more papers by this author and Anthony A. Bavry, MD, MPHFrom University of Florida, Gainesville, FL 32610.Search for more papers by this authorAuthor, Article, and Disclosure Informationhttps://doi.org/10.7326/0003-4819-154-5-201103010-00017 SectionsAboutFull TextPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinkedInRedditEmail IN RESPONSE:We appreciate the comments from Dr. Leclercq and colleagues about our letter on limitations of MEDLINE. The authors elucidate the central point of our letter, which is that incomplete MeSH indexing can contribute to inaccurate searches when limits are used. To clarify our search strategy, the precise search string that we used was (“Platelet Glycoprotein GPIIb-IIIa Complex”[MeSH]) OR eptifibatide OR abciximab OR tirofiban. On repeating this search with the publication date limit of 1 February 2010 and the search limit “clinical trial,” we found the same results as Dr. Leclercq and colleagues: 1003 records.After discussion with researchers ... Author, Article, and Disclosure InformationAffiliations: From University of Florida, Gainesville, FL 32610.Disclosures: None disclosed. PreviousarticleNextarticle Advertisement FiguresReferencesRelatedDetailsSee AlsoLimitations of the MEDLINE Database in Constructing Meta-analyses David E. Winchester and Anthony A. Bavry Limitations of the MEDLINE Database in Constructing Meta-analyses Edith Leclercq , Bianca Kramer , and Winnie Schats Metrics 1 March 2011Volume 154, Issue 5Page: 372KeywordsClinical trialsConflicts of interestDatabasesGlycoproteinsLibrariesPlatelets ePublished: 1 March 2011 Issue Published: 1 March 2011 CopyrightCopyright © 2011 by American College of Physicians. All Rights Reserved.PDF DownloadLoading ...

Calcium mobilization by the plant estrogen ferutinin does not induce blood platelet aggregation

Platelet activation is closely associated with an increase in intracellular Ca2+ concentration. Various compounds including Ca2+ ionophores are able to trigger platelet aggregation by increasing intracellular Ca2+ concentration in platelets. In the present study, we monitored the effect of the phytoestrogen ferutinin, which acts as a Ca2+ ionophore in human blood platelets; its ionophore-like properties include upregulation of [Ca2+]in, activation of fibrinogen receptors and increased fibrinogen binding. Using spectrofluorometry and triple-color flow cytometry, we demonstrate that ferutinin increases [Ca ] in both isolated platelets and platelets in whole blood from humans. This effect was almost completely blocked by the Ca2+ chelator EGTA and was not sensitive to either Gd3+ or econazole, which inhibit VOC and SOC channels, respectively. Nor was the effect sensitive to thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ ATPases. Ferutinin stimulated the expression of the active form of the GPIIb-IIIa complex and whole blood platelet aggregation only weakly and had no statistically significant effect on the binding of fibrinogen. These results demonstrate apparently inconsistent effects of ferutinin, which raises intraplatelet Ca2+ concentration but fails to have an effect on spontaneous blood platelet aggregation. This pattern of responses may be caused by the combination of ferutinin’s Ca2+ ionophoric and estrogenic properties.

Relative Efficacy of Steroid Therapy in Ameliorating Autoimmune Thrombocytopenia Mediated by Anti-Platelet GPIIbIIIa Versus GPIbα Antibodies.

Abstract 1323Poster Board I-345 BackgroundIdiopathic thrombocytopenic purpura (ITP) is a common autoimmune disease characterized by autoantibody-induced platelet destruction and consequent bleeding disorders. Despite considerable investigation, the pathogenesis of ITP remains incompletely understood, and we currently lack a consensus for therapy. The major targeted platelet autoantigens are located on two distinctive platelet receptors; the GPIIbIIIa and GPIb complexes. It has been reported that thrombocytopenia induced by anti-GPIba antibodies, which may differ from that induced by anti-GPIIbIIIa, can be mediated via an Fc-independent pathway. We further demonstrated in a murine model that anti-GPIba-mediated-ITP is less responsive to IVIG treatment (Blood. 2006; 108(3):943-6), which is consistent with a subsequent retrospective study in human ITP patients. Since IVIG is expensive and steroid therapy is the first line of treatment for most patients, particularly in developing countries, we tested whether gluococorticosteroid therapy has equal efficacy in ITP patients caused by these two different antibodies. Patients and MethodsA total of 107 ITP patients (71 females and 36 males) were recruited for the present study. Patients were first treated with Dexamethasone (DXM, 20mg/day x 3-5 days), then changed to oral administration of Prednisone (1mg/kg/day). The dose of Prednisone was gradually decreased and maintained for = 30 days. The patients were considered as responders to the therapy if no obvious haemorrhage was detected and their platelet counts were raised to = 50 × 109/L or increased by = 30 × 109/L from their original platelet count before treatment. All patients' plasma samples were collected before therapy and specific autoantibodies against GPIIbIIIa and/or the GPIb complex were measured with a monoclonal antibody immobilization of platelet antigen assay (MAIPA). ResultsAmong the 107 patients, 23% (25/107) had antibodies against GPIIbIIIa, 18% (19/107) had antibodies against the GPIb complex, 34% (36/107) had antibodies against both the GPIIbIIIa and GPIb complex, and 25% (27/107) had no detectable antibody to either of these two antigens. We found that a majority of the patients with antibodies against GPIIbIIIa (15/22) were sensitive to the steroid therapy, which is significantly different from those with antibodies against the GPIb complex (5/19) (X2 = 7.15, P <0.01) or those with antibodies against both the GPIb complex and GPIIbIIIa (6/34) (X2 = 14.5, P <0.01). Although no statistically significant difference was observed between patients with anti-GPIb antibodies and those with both anti-GPIb and anti-GPIIbIIIa antibodies (X2 = 0.55, P >0.05), there is a tendency that patients with double positive antibodies were less responsive to steroid therapy therapy. The most sensitive patients for this therapy were those without detectable antibodies to either of these two autoantigens (21/27). ConclusionsOur data suggest that anti-GPIb-mediated ITP may be less responsive to both IVIG and steroid therapies when compared to anti-GPIIbIIIa-mediated ITP. The pathogenesis and treatment of anti-GPIb-mediated ITP merits further investigation. DisclosuresNo relevant conflicts of interest to declare.

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.

Analysis of phenotype and genotype in eight Chinese pedigrees with glanzmann thrombastheuia

Objective To study the GPⅡb/Ⅲa gene mutations of eight glanzmann thrombasthenia(GT) pedigrees. Methods Responses of eight probands to different agonists were observed by platelet aggregation test and the amount of αⅡb and β3 was determined by flow cytometry. All the exons of Ⅱb and Ⅲ a genes were amplified by PCR followed by sequencing for mutational screening. Further analysis of the normal population excluded the possibility of mutational sites as a polymorphism. Results Eight probands showed normal PLT counts, dispersion of the platelet particles without aggregation, prolonged bleeding time and severely reduced platelet aggregation in response to the physiological agonists- ADP, epinephrine, and collagen, but relatively normal aggregation of PLT in response to ristocetin. Flow cytometry showed that all probande were Ⅰ type GT, except that proband 2 was Ⅲ type GT and proband 6 was Ⅱ type GT. The sequencing results showed that twelve different types mutations were present in eight probands, including GIOA, Gl412T, GII99A, 1525deiC, G2223T, C2671T, 2930delG, IVSI5 (-1) delG, A2334C, C1750T, 69-79del and CA70A. We were not able to detected any mutations in GP Ⅱb/Ⅲa gone on proband 3. Conclusions GT is mainly caused by GPⅡb/Ⅲa gene mutations. G10A, 69-79del, C470A,G1412T, G2223T, C2671T and 1525delC were the novel mutations causing GT. Key words: Thrombasthenia; Platelet glycoprotein GPⅡb-Ⅲa complex; Genotype; Phenotype

HoxA2 Regulates Proliferation of an Embryonic Megakaryocyte Progenitor, Which Can Effectively Produce Platelets In Vitro.

Megakaryocytes are rare cells in the bone marrow, and are solely responsible for thrombopoiesis throughout the life of the organism. Various transcription factors regulating megakaryopoiesis have been identified, including GATA1, Fli1 and NF-E2, which are important for the maturation and differentiation of megakaryocytes and SCL and FOG1, which play important roles in the specification and proliferation of the early megakaryocyte progenitor but no single factor capable of sustaining long-term proliferation of the megakaryocyte progenitor has been identified. The rarity of this progenitor, and the difficulty of proliferating megakaryocytes in vitro have seriously hampered the effort to produce large quantities platelets in vitro, and to date the only source of platelets for transplantation remains human donors. In this study, through a gain of function screen of Hox genes in which each paralog group was tested, we identified HoxA2 as an important transcription factor for the proliferation of early embryonic megakaryocytic progenitors. HoxA2 promoted the outgrowth of undifferentiated megakaryocyte progenitors when transduced into precirculation yolk sac stem and progenitor cells, however it had no effect on megakaryocytic progenitors derived from definitive HSCs of adult bone marrow. To study this unique embryonic regulatory circuit further, we generated an inducible mouse embryonic stem cell line, in which the expression of murine HoxA2 is regulated by doxycycline. Embryoid body differentiation of these cells, followed by sorting and reculture of the Kit+/CD41+ hematopoietic progenitor fraction at day 6 in the presence of continual HoxA2 expression allowed undifferentiated c-Kit+/CD41+/CD45+ megakaryocyte progenitors to proliferate for upwards of 2 months. When doxycycline is removed, numerous gene expression changes take plase, and these progenitors differentiate into mature c-Kitneg/CD41high/CD45neg megakaryocytes, which undergo proplatelet formation, releasing large quantities of platelets (∼107/ml) into the medium. As this rate of production can be sustained for weeks, a highly efficient platelet bioreactor is enabled. The platelets express the integrin αIIb-β3 (GPIIb-IIIa complex), and upon stimulation they bind fibrinogen, demonstrating functionality. We are currently investigating the transplantation potential of these cells.Our data suggest that adult and embryonic megakaryopoiesis have distinct regulatory circuits, and demonstrate a novel and uniquely embryonic role for HoxA2, which opens up the possibility of producing industrial quantities of platelets in vitro for therapeutic purposes.

P.152 Acupuncture treatment of purulent-inflammatory processes in maxillofacial area

Preincubation of human platelets with two pairs of immunologically identical monoclonal antibodies (mab) directed against epitopes on the membrane glycoprotein IIb-IIIa complex - two of them (Gi3 and Gi5) precipitating glycoproteins IIb-IIIa, and two (Gi4 and Gi6) recognizing glycoprotein IIIa -induced slightly different inhibitory effects on platelet function. Gi3 and Gi5 blocked platelet aggregation and 14C-serotonin release from platelets induced by ADP, collagen, thrombin, arachidonic acid, ionophore A 23187, platelet activating factor and ristocetin (in the presence of divalent ions). Ristocetin-induced platelet agglutination (in the presence of EDTA) was not inhibited by these mab. Gi4 had no effect on platelet aggregation and release. Gi6 had the same effect on platelet function as Gi3 and Gi5, but only when incubated with Zw(a) (PIA1)-positive platelets with the exception that collagen-induced aggregation was not affected. We conclude that the similar, though not identical, effects on platelet function induced by mab against different epitopes on the glycoprotein complex IIb-IIIa indicate an “unspecific” interference with platelet membrane function possibly due to steric hindrance by mab of the fibrinogen receptor or by inhibition of conformational changes in the GP IIb-IIIa complex necessary for exposure of the fibrinogen binding site.

Acetylsalicylic acid is compounding to antiplatelet effect of C-reactive protein

The contribution of inflammatory process to the modulation of platelet response to acetylsalicylic acid (ASA) remains obscure. In our study, we examined the in vitro effect of C-reactive protein (CRP) on the ASA-mediated inhibition of collagen-stimulated platelet reactivity. Influence of CRP on platelet responsiveness to ASA was analysed using classical turbidimetric aggregation and flow cytometry. When acting alone, both C-reactive protein and ASA inhibited collagen-dependent platelet aggregation and reduced the expressions of two platelet surface membrane activation markers: P-selectin and activated GPIIbIIIa complex. Compared to the effects observed for ASA alone, the simultaneous action of both agents lead to further reductions in platelet aggregation (by 56.7 ± 1.0% vs. 14.9 ± 0.6%, p < 0.0001) and lowered the expressions of platelet surface membrane P-selectin (by 72.1 ± 5.3% vs. 65.0 ± 6.0%, p < 0.01) and activated GPIIbIIIa (by 67.0 ± 5.6% vs. 47.7 ± 8.3%, p < 0.01). In general, our findings showed for the first time the augmenting effect of native C-reactive protein in the antiplatelet action of acetylsalicylic acid. Thus, we conclude that the effectiveness of aspirin therapy may strongly depend upon the presence of native CRP in circulation.

Mutations in GPIIIa molecule as a cause for Glanzmann thrombasthenia in Indian patients

Glanzmann thrombasthenia (GT) results from a quantitative or qualitative defect of GPIIb-IIIa complex, the fibrinogen receptor on platelets, which plays a very important role in platelet aggregation. In this report we describe the molecular studies on 22 patients with Glanzmann Thrombasthenia at our institute. The main objective was to identify the mutations present in our GT population in order to establish a strategy for genetic counseling and antenatal diagnosis. Twenty-two patients with GT were included in the present study. Complete blood count (CBC), platelet aggregation, flow cytometry, Western blot, single strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) were performed in all the patients. The patients showing an abnormal migration pattern in SSCP or DGGE were sequenced further on an automated sequencer. Of the 22 patients studied, mutations were detected in 12 individuals. Of these, 11 were novel mutations and one mutation Y115C was reported earlier. Flow cytometric analysis showed the absence of receptors in type I GT, highly reduced levels in type II GT and normal levels in type III GT. The DGGE analysis and SSCP analysis of the patients showed different migration patterns. Sequencing was performed in all patients showing an abnormal migration pattern. Of the 22 cases studied mutations could be detected in 12 cases of GT. We could detect six patients with point mutations, four patients with insertions and five patients with deletion mutations. Exon 4 has been found to be the most common site for mutations in our patients. This study has shown a wide array of mutations present in our GT patients which would be extremely useful in genetic counseling and prenatal diagnosis, essential in preventing these disorders in succeeding generations.

Decreased Platelet Expression of MYL9 (Myosin Regulatory Light Chain Polypeptide) and Other Genes with Platelets Dysfunction and CBFA2 Mutation: Insights from Platelet Expression Profiling.

We have previously reported (Blood , 87:1368, 1996) a patient with a mild thrombocytopenia, and markedly abnormal aggregation, secretion, phosphorylation of pleckstrin and myosin light chain (MLC), and activation of GPIIb-IIIa complex. We recently showed (Blood 2004, 103: 948–954) in this patient a heterozygous mutation in hematopoietic transcription factor CBFA2 (Core Binding factor A2, also called AML1) and decreased platelet PKC-theta. We postulated that platelet expression profiling would provide insights into the pathophysiological mechanisms and gene expression changes resulting from a CBFA2 mutation. We performed platelet expression profiling in 4 control subjects (6 profiles, 2 individuals profiled twice) and the patient (profiled twice, 10 months apart) using the Affymetrix microarray Human U133A and B GeneChips (total ~44000 probe sets). ~10,000 probe sets (genes and ESTs) are expressed in human platelets. Iterative comparisons of data from each individual platelet profiles had correlation coefficients (r) > 0.85. Using GeneSpring® analytical software we selected genes that showed at least 1 present call in the 8 profiles, and p<0.05 showing a significant difference between the 2 groups. Relative expression values <0.5 and >2 were considered as significant changes. In the patient, 298 probe sets were significantly down regulated and 29 significantly upregulated. We focused on genes showing a > 5-fold decrease (fold change, fc < = 0.20). There was a striking ~75 fold decrease (fc = 0.013; p=0.00022) in myosin regulatory light chain polypeptide (MYL9 gene). Other down-regulated genes included calcium binding protein 5 (CABP5, fc = 0.02; a protein with similarity to calmodulin), Na/K/Ca exchanger member 3 (SLC24A3, fc = 0.11), b3-endonexin (ITGB3BP, fc = 0.01), platelet factor 4 variant 1 (PF4V1, fc = 0.03), chemokine CXCL5 (ENA-78) (fc = 0.20), tropomyosin I (alpha) (TPM1, fc = 0.13) and arachidonate 12-lipoxygenase (ALOX-12, fc = 0.22). The expression array findings were validated by decreased platelet mRNA levels on real time PCR or by immunoblotting (platelet PF4). Several down-regulated genes are directly relevant to the patient's platelet defect. Agonist-stimulated MLC phosphorylation is impaired in patient platelets. The strikingly decreased expression of MYL9 with normal expression of MLC kinase (MLCK, by array and real time PCR) suggests that the blunted myosin phosphorylation arises from decreased MYL9 than due to decreased MLCK. Myosin light chains play a role in diverse processes including in cell contractility, mobility and cytokinesis, and are relevant to impaired platelet function and production. The decreased expression of the Na/K/Ca exchanger and b3-endonexin 3 (both implicated in activation of GPIIb-IIIa) provide mechanisms for the diminished GPIIb-IIIa activation. Chemokine CXCL5 and PF4V1 are expressed in megakaryocytes, and the genes are colocalized to “chemokine cluster” on chromosome 4. 12-lipoxygenase, a major platelet enzyme, regulates arachidonic acid metabolism and production of the 12-hydroxy eicosanoids. This is the first proof of concept that platelet expression profiling can be applied to obtain new insights into the molecular basis of inherited platelet dysfunction. Further studies will provide new information on the genes regulated by CBFA2, and on mechanisms regulating platelet production and activation.

Procoagulant potential of platelet α granules

In this brief review of the literature it is pointed out that during platelet activation and degranulation platelet α granules leave the platelet interior through blebs in platelet plasma membrane and through the tips of the pseudopods, and then accumulate in the external milieu. There they undergo disintegration and secondary adhesion to the platelet plasma membranes. During their disintegration they expose their tightly packed GPIIb–IIIa complexes, annexin V stainable aminophospholipids, factor V, and the membrane markers CD62 and CD63. There is also demasking of lysosomal acid phosphatase activity and microvesicle formation. Lysosomal nature of platelet α granules is mentioned. It is suggested that platelet α granules are the sole source of platelet procoagulant activity and platelet microparticles (MP) or microvesicles (MV). The implications of this concept for antiplatelet therapy are discussed. A relationship of this process to tissue factor exposure and apoptosis is suggested.

Association of CBFA2 mutation with decreased platelet PKC-θ and impaired receptor-mediated activation of GPIIb-IIIa and pleckstrin phosphorylation: proteins regulated by CBFA2 play a role in GPIIb-IIIa activation

AbstractThe mechanisms by which agonists activate glycoprotein (GP) IIb-IIIa function remain unclear. We have reported data on a patient with thrombocytopenia and impaired receptor-mediated aggregation, phosphorylation of pleckstrin (a protein kinase C [PKC] substrate), and activation of the GPIIb-IIIa complex. Abnormalities in hematopoietic transcription factors have been associated with thrombocytopenia and platelet dysfunction. To define the molecular mechanisms, we amplified from patient platelet RNA exons 3 to 6 of core-binding factor A2 (CBFA2) cDNA, which encompasses the DNA-binding Runt domain; a 13-nucleotide (nt) deletion was found (796-808 nt). The gDNA revealed a heterozygous mutation (G>T) in intron 3 at the splice acceptor site for exon 4, leading to a frameshift with premature termination in the Runt domain. On immunoblotting, platelet CBFA2, PKC-θ, albumin, and IgG were decreased, but pleckstrin, PKC-α, -βI, -βII, -η, -ϵ, -δ, and -ζ, and fibrinogen were normal. Our conclusions are that (1) CBFA2 mutation is associated with not only thrombocytopenia, but also impaired platelet protein phosphorylation and GPIIb-IIIa activation; (2) proteins regulated by CBFA2 are required for inside-out signal transduction-dependent activation of GPIIb-IIIa; and (3) we have documented the first deficiency of a human PKC isozyme (PKC-θ), suggesting a major role of this isozyme in platelet production and function. (Blood. 2004;103:948-954)

Simultaneous isolation of platelet factor 4 and glycoprotein IIb–IIIa complex from rabbit platelets, and characterization of specific chicken antibodies to assay them

Rabbits are frequently used as models for studying coagulation and platelet disorders. However, few reports on literature have dealt with the purification and characterization of rabbit platelet proteins. Herein a protocol for the simultaneous purification of rabbit platelet factor 4 (PF4) and platelet glycoprotein IIb–IIIa (GPIIb–IIIa, integrin α IIbβ 3) is described. Specific antibodies were raised in laying chicken, which were used for assaying PF4 by ELISA, and GPIIb–IIIa by direct immunofluorescence and flow cytometry. Furthermore, the binding of monoclonal antibodies specific for GPIIb–IIIa complex (P2), ligand-induced binding site of GPIIIa (LIBS1) and rabbit P-selectin (12A7), as well as of polyclonal IgY specific for rabbit GPIIb–IIIa, was compared in quiescent and thrombin-activated platelets. Polyclonal anti-rabbit PF4 IgY was a specific and sensitive probe that could be used for assaying PF4 in plasma samples. GPIIb–IIIa expression was increased in thrombin-activated platelets, as evaluated by flow cytometric analysis using P2 and polyclonal antibodies raised in chickens. Rabbit GPIIb–IIIa also exhibited a conformational modification that caused the appearance of ligand-induced binding sites. Increased P-selectin expression, used as a positive control, was also noticeable in thrombin-activated platelets. These data evidence that antibodies raised in laying chickens specific to rabbit PF4 and GPIIb–IIIa, as well as certain monoclonal antibodies specific for human GPIIb–IIIa, may be used for investigating rabbit platelet physiology.