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Post-transfusion purpura: case report.

Post-transfusion purpura (PTP) is a rare bleeding disorder of platelet alloimmunization that perhaps occurs as an anamnestic reaction. Most commonly, it is observed in PlA1-negative subjects previously sensitized with PlA1 platelet antigen either through PlA1-positive pregnancy or PlA1-positive transfusion. PTP appears with sudden severe thrombocytopenia, purpura, and often life-threatening hemorrhage within 5-10 days of blood transfusion. It is believed to be self-resolving. Yet inactivity risking dangerous bleeding can be disastrous. Treatment with intravenous immunoglobulin, corticosteroids, exchange transfusion, and plasmapheresis has been reported with variable success. No single modality, however, is effective in all cases. Not more than 150 cases of PTP seem to have been reported. We present two such cases. Both were multiparous PlA1-negative women given a blood transfusion for the first time. Corticosteroid therapy failed in both. One responded to intravenous immunoglobulin, while for the other plasmapheresis was the only life-saving modality. One of them subsequently required a blood transfusion for surgical intervention, which could be given uneventfully.

Adult↗

Intense exercise increases shear-induced platelet aggregation in men through enhancement of von Willbrand factor binding, glycoprotein IIb/IIIa activation, and P-selectin expression on platelets.

Vigorous exercise transiently enhances the risk of primary cardiac arrest. Shear-induced platelet aggregation (SIPA) is an important mechanism in arterial thrombogenesis. This study investigates whether intense exercise affects SIPA, and elucidates mechanisms that underlie SIPA. Eighteen sedentary healthy men engaged in intense exercise (about 80% of maximal oxygen consumption) for 40 min on a bicycle ergometer. Platelet aggregation, binding of von Willebrand factor (vWF) to platelets, and activation of glycoprotein (GP) IIb/IIIa and expression of P-selectin on platelets induced by shear stress were analyzed both before and immediately after exercise. Analytical results demonstrated that: (1). the levels of plasma vWF antigen and activity were enhanced after intense exercise, (2). intense exercise increased either shear- or ristocetin-induced platelet aggregation and was accompanied by an increase in vWF binding to platelets and vWF-mediated GP IIb/IIIa activation at high shear flow, and (3). shear-induced P-selectin expression in the absence or the presence of exogenous vWF was enhanced by intense exercise. Therefore, we conclude that intense exercise promotes the extent of SIPA, possibly by enhancing the ability of vWF to bind to platelets and the subsequent activation of GP IIb/IIIa complexes, as well as the expression of P-selectin in response to shear stress, which in turn may augment the risk of vascular thrombosis.

Adult↗

Evidence for epistasis between SLC6A4 and ITGB3 in autism etiology and in the determination of platelet serotonin levels.

Autism is a neurodevelopmental disorder of unclear etiology. The consistent finding of platelet hyperserotonemia in a proportion of patients and its heritability within affected families suggest that genes involved in the serotonin system play a role in this disorder. The role in autism etiology of seven candidate genes in the serotonin metabolic and neurotransmission pathways and mapping to autism linkage regions (SLC6A4, HTR1A, HTR1D, HTR2A, HTR5A, TPH1 and ITGB3) was analyzed in a sample of 186 nuclear families. The impact of interactions among these genes in autism was assessed using the multifactor-dimensionality reduction (MDR) method in 186 patients and 181 controls. We further evaluated whether the effect of specific gene variants or gene interactions associated with autism etiology might be mediated by their influence on serotonin levels, using the quantitative transmission disequilibrium test (QTDT) and the restricted partition method (RPM), in a sample of 109 autistic children. We report a significant main effect of the HTR5A gene in autism (P = 0.0088), and a significant three-locus model comprising a synergistic interaction between the ITGB3 and SLC6A4 genes with an additive effect of HTR5A (P < 0.0010). In addition to the previously reported contribution of SLC6A4, we found significant associations of ITGB3 haplotypes with serotonin level distribution (P = 0.0163). The most significant models contributing to serotonin distribution were found for interactions between TPH1 rs4537731 and SLC6A4 haplotypes (P = 0.002) and between HTR1D rs6300 and SLC6A4 haplotypes (P = 0.013). In addition to the significant independent effects, evidence for interaction between SLC6A4 and ITGB3 markers was also found. The overall results implicate SLC6A4 and ITGB3 gene interactions in autism etiology and in serotonin level determination, providing evidence for a common underlying genetic mechanism and a molecular explanation for the association of platelet hyperserotonemia with autism.

Adolescent↗

Cyclosporin A does not affect platelets in children with idiopathic nephrotic syndrome.

The immunosuppressive agents administered to maintain the remission of idiopathic nephrotic syndrome (INS) may have a deleterious effect on several cell types. The aim of this study was to analyze platelet activation and reactivity in children with INS treated with cyclosporin A (CyA). The study groups comprised 16 children with remission of INS induced by CyA and 16 children with glucocorticosteroid-induced remission 8 weeks from the onset of INS relapse. Fifteen healthy children served as controls. Surface expression of CD61, CD62P, and CD42b on resting and thrombin-stimulated platelets was analyzed with flow cytometry. No differences between groups were found in CD61, CD62P, and CD42b surface expression, but markers of the coagulation cascade and fibrinolysis or endothelial injury (F1+2 prothrombin fragments, tissue plasminogen activator inhibitor 1) were elevated in patients treated with CyA compared with children on steroids and healthy controls. No correlations between markers of platelet function and CyA concentration were found. We postulate that CyA administration in nephrotic patients causes an activation of thrombinogenesis but does not influence platelet activation and reactivity in INS.

Adolescent↗

New method for DNA microarrays development: applied to human platelet antigens polymorphisms.

DNA microarrays are a powerful experimental tool for the detection of specific genomic sequences and are invaluable to a broad array of applications: clinical diagnosis, personalized medicine, drug research and development, gene therapy, food control technologies, and environmental sciences. Alloimmunization to human platelet antigens (HPAs) is commonly responsible for neonatal alloimmune thrombocytopenia, post-transfusional purpura and platelet transfusion refractoriness. Using DNA microarrays, we developed a diagnosis to type the biallelic HPA-1 platelet group. The region for the human genomic DNA sequence that contains the polymorphism responsible for HPA-1 alleles was amplified by polymerase chain reaction (PCR). The expected DNA fragments were hybridized on DNA microarrays, and the data were analyzed using specially developed software. Our initial results show that the two HPA-1 antigens polymorphisms containing a single base difference were detected using DNA microarrays.

Antigens, Human Platelet↗

Integrin alpha-2 and beta-3 gene polymorphisms and breast cancer risk.

Integrins are cell surface receptors, which mediate cell-to-cell and cell-to-extracellular matrix adhesion. Some of them, e.g. alpha(V)beta(3), alpha(IIb)beta(3) and alpha(2)beta(1), have been suggested as key players for cancer development and tumor metastasis. Two polymorphisms in the gene for the alpha(2) component, ITGA2 807C>T and 1648G>A, have been associated with the cell-surface density of integrin alpha(2)beta(1). The 176T>C polymorphism in the ITGB3 gene, encoding the beta(3) subunit of integrins alpha(IIb)beta(3) and alpha(V)beta(3), modifies a variety of traits of beta(3) expressing cells. To analyze the role of ITGA2 and ITGB3 polymorphisms for breast cancer risk and prognosis, we performed a case-control study including 500 female breast cancer patients and 500 healthy female age-matched control subjects. All study participants were of Caucasian origin (Austria, Middle-Europe). The ITGA2 1648_AA genotype was significantly associated with breast cancer (odds ratio 3.12; 95% confidence interval 1.11-8.77). Carriers of the most common ITGA2 haplotype (807C_1648G, 'wildtype') were at decreased risk for breast cancer (odds ratio 0.72; 95% confidence interval 0.53-0.98). A histological grade of 3 or 4 was found more often in ITGA2 807TT subjects (p=0.039 compared to CC+CT genotypes) and carriers of an ITGA2 1648A allele (p=0.017 compared to GG genotype). Carriers of the ITGA2 807C_1648G haplotype were less likely to have a histological grade 3 or 4 compared to non-carriers (p=0.003). The ITGB3 176T>C polymorphisms was not associated with breast cancer susceptibility. In a Cox-regression analysis, carriers of the homozygous ITGB3 176-CC genotype had a higher risk for metastasis (relative risk 2.3; 95% CI 1.3-4.2; p=0.005). We conclude that functional polymorphisms in integrin genes ITGA2 and ITGB3 influence the development and progression of breast cancer, respectively. The precise mechanism remains to be determined, but likely involves dysregulated signaling pathways.

Breast↗

Molecular cloning and expression of glycoprotein IIb and IIIa.

OBJECTIVE: To amplify the cDNA genes of GPIIb, GPIIIa, then construct the eukaryotic expression carriers of GPIIb and GPIIIa respectively, finally establish CHO cell lines stably expressing GPIIb and GPIIIa. METHODS: Human erythroleukemia (HEL) cells were cultured for total RNA extraction. RT-PCR was accomplished using the specific GPIIb, GPIIIa primers designed according to Genbank by Primer 5, then each of cDNAs were obtained. The expressive vector pcDNA3.1(+) and PCR products were cut by NheI and HindIII, and then the fragements were directly cloned to pcDNA3.1(+) because of having the same adhesive ends. Then pcDNA3.1(+)IIb and pcDNA3.1(+)IIIa were transfected into CHO cells respectively by Lipofectamine 2000. The cell lines expressing GPIIb, GPIIIa were screened by G418. Then the Chinese hamster ovary (CHO) cell lines were examed through flow cytometry (FCM) and RT-PCR to detect the expression of GPIIb, GPIIIa in CHO cells. RESULTS: The cDNAs of GPIIb and GPIIIa were amplidied by RT-PCR, and the pcDNA3.1(+)IIb and pcDNA3.1(+)IIIa were constructed respectively. By sequencing and double digestion, pcDNA3.1(+)IIb and pcDNA3.1(+)IIIa were all correct. Expression of GPIIb and GPIIIa were detected on transfected CHO cells by FCM and RT-PCR. CONCLUSIONS: (1) Succeeded in constructing pcDNA3.1(+)IIb, pcDNA3.1(+)IIIa. (2) Succeeded in getting the cell lines expressing GPIIb, GPIIIa.

Animals↗

The effect of glycoprotein IIIa PIA 1/A2 polymorphism on the PFA-100 response to GP IIb IIa receptor inhibitors-the importance of anticoagulants used.

Antithrombotic drugs including glycoprotein (GP) IIb IIIa receptor inhibitors have significantly reduced ischaemic events in coronary disease. Variability in the response to GP receptor inhibitors has been observed both with healthy individuals and in clinical studies. One single nucleotide polymorphism on GP IIIa (PI(A1/A2)) correlates with increased risk for cardiovascular events in many studies. In this study we investigated whether this polymorphism associates with individual differences in the response to GP IIb IIIa receptor inhibitors in healthy individuals. Fresh blood samples were collected randomly from individuals without a history of coronary disease. Blood samples were anticoagulated with either sodium citrate or with PPACK. The ability of different GP IIb IIIa receptor inhibitors (tirofiban, eptifibatide and abciximab) to inhibit platelet aggregation was investigated using a commercial PFA-100 analyser. At baseline, the function of platelets with different PI(A) genotypes did not differ from each other. With sodium citrate anticoagulated samples, tirofiban prolonged the closure time slightly more rapidly when platelets with PI(A 2 A 2) genotype were used than with other genotypes (p<0.05) both on epinephrine-collagen and ADP-collagen coated membranes. With eptifibatide or abciximab no differences were observed. If an anticoagulant not affecting Ca(2+) concentration (PPACK) was used, no differences were observed between different GP IIIa genotypes and the ability of any of the GP IIb IIIa receptor inhibitors to prolong the closure time. The effect of tirofiban and eptifibatide was significantly affected by the anticoagulant used (p<0.001), whereas abciximab functioned equally regardless of the anticoagulant. Glycoprotein IIIa PI(A2) allele has been found in many studies to associate with risk of thrombosis. In healthy controls the function of GP IIb IIIa receptor inhibitors on platelets with different PI(A) genotypes was modified by anticoagulants used. PI(A) polymorphism does not explain the interindividual variance in the response to GP IIb IIIa receptor inhibitors in healthy individuals. The study, however, demonstrates the importance of the anticoagulant used in in vitro studies and the important role of Ca(2+) concentration with tirofiban and eptifibatide, but not with abciximab.

Adult↗

A comprehensive analysis of 12 thrombophilic mutations and related parameters in patients with inflammatory bowel disease: data from Turkey.

BACKGROUND: Possible association of inflammatory bowel disease (IBD) with the most common inherited prothrombotic conditions has been the focus of many investigations. Advance in modern molecular biology is expanding the thrombophilia evaluation steadily. We tried to put forward a comprehensive thrombophilic profile in IBD and to see the probable role of this profile in pathogenesis. METHODS: A total of 60 adults (33 patients and 27 healthy controls) were included. We used the CVD-StripAssay which is based on the reverse-hybridization principle to identify a total of 12 thrombophilic gene mutations: Factor V R506Q, Factor V H1299R, prothrombin G20210A, Factor XIII V34L, beta-Fibrinogen-455 G-A, PAI-1 4G/5G, platelet GPIIIa L33P, MTHFR C677T, MTHFR A1298C, ACE I/D, Apo B R3500Q and Apo E2/E3/E4, respectively. Besides, we evaluated many related blood parameters such as protein C, protein S, AT-III, IL-6, TNF-alpha, Apo-A1, Apo-B100, homocysteine (tHcy) etc. using commercially available assays. RESULTS: The frequencies of genetic polymorphisms were found to be statistically insignificant among patients and controls, except for three: Beta-Fibrinogen-455G-A, MTHFR A1298C and ACE-I/D. Two patients with a history of deep venous thrombosis had more than one polymorphism. Patients with MTHFR C677T and MTHFR A1298C gene mutations had a similar mean tHcy levels with controls. Patients with Apolipoprotein B R3500Q and Apolipoprotein E4 gene mutations had similar mean LDL-cholesterol levels. Mean total cholesterol and triglyceride levels were similar in patients and controls of Apo E2, E3, E4 alleles. CONCLUSION: Predominantly, the presence of genetic mutations that predispose to hypercoagulable states does not appear to be in correlation with IBD. There was a statistical difference between the proportions of the mutated allele frequencies of Beta-Fibrinogen-455G-A, MTHFR A1298C and ACE-I/D in IBD.

Adolescent↗

Inhibitory mechanisms of low concentrations of oxidized low-density lipoprotein on platelet aggregation.

The intracellular mechanisms underlying oxidized low-density lipoprotein (oxLDL)-signaling pathways in platelets are not yet completely understood. Therefore, the aim of this study was to further examine the effects of oxLDL in prevention of platelet aggregation. In this study, oxLDL concentration-dependently (40-120 microg/ml) inhibited platelet aggregation in human platelet-rich plasma stimulated by agonists. Moreover, oxLDL (40 and 80 microg/ml) markedly decreased the fluorescence intensity of platelet membranes tagged with diphenylhexatriene. Rapid phosphorylation of a protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by PDBu (150 nM). This phosphorylation was markedly inhibited by oxLDL (40 and 80 microg/ml) in phosphorus-32-labeled platelets. In addition, oxLDL (40 and 80 microg/ml) markedly increased levels of cyclic AMP and cyclic AMP-induced vasodilator-stimulated phosphoprotein (VASP) Ser(157) phosphorylation. The thrombin-evoked increase in pHi was inhibited in the presence of oxLDL (40 and 80 microg/ml). These results indicate that the antiplatelet activity of oxLDL may involve the following pathways. (1) oxLDL may initially induce conformational changes in platelet membranes, leading to inhibition of the activation of protein kinase C, followed by inhibition of P47 protein phosphorylation, and intracellular Ca(2+) mobilization. (2) oxLDL also activated formation of cyclic AMP and cyclic AMP-induced VASP Ser(157) phosphorylation, resulting in inhibition of the Na(+)/H(+)exchanger; this leads to reduced intracellular Ca(2+) mobilization, and ultimately to inhibition of platelet aggregation. This study further provides new insights concerning the effects of low concentrations of oxLDL on platelet aggregation.

Calcium↗

Alloimmune thrombocytopenia: fetal and neonatal losses related to cordocentesis.

OBJECTIVE: This report describes the increased risks of cordocentesis in fetuses affected with alloimmune thrombocytopenia. STUDY DESIGN: As part of a multicenter treatment study clinical and laboratory data from five pregnancies with alloimmune thrombocytopenia in which there was a fetal or neonatal loss associated with cordocentesis were reviewed. The fetal or neonatal deaths were all thought to be a result of exsanguination. These fetuses were compared with a group of 44 affected fetuses who underwent the same procedure but who survived. The data were analyzed by the Wilcoxon rank-sum test and the two-tailed Fisher's exact test. A p value < 0.05 was considered significant. RESULTS: The mean platelet count at cordocentesis was significantly lower in the cases than in the controls (5.8 vs 32.8 x 10(9)/L, p = 0.005). The incidence of antenatal intracranial hemorrhage in the untreated sibling of the prior affected pregnancy was significantly greater in the cases than in the controls (two of five vs one of 42, p = 0.02). CONCLUSION: Fetuses affected with alloimmune thrombocytopenia are at increased risk for fatal exsanguination associated with cordocentesis.

Antigens, Human Platelet↗

Identification of PlAl alloantigen domain on a 66 kDa protein derived from glycoprotein IIIa of human platelets.

Incubation of platelets with chymotryptin leads to the exposure of fibrinogen receptors and to the appearance of a 66 kDa membrane component on the surface of platelets. Both glycoprotein IIIa (GP IIIa) and a 66 kDa component were precipitated from detergent extracts of solubilized, surface radiolabeled chymotrypsin-treated platelets by human anti-PlAl antisera. Moreover, the presence of the P1A1 antigen was identified on GP IIIa (but not on GP IIb) and on a 66 kDa protein by means of immunoblot procedures using platelet Triton X-114 extracts and these purified proteins. Anti-PlAl antiserum did not recognize GP IIIa on the surface of intact (untreated) platelets nor the 66 kDa protein on the surface of chymotrypsin-treated platelets of PlAl-negative individuals. The present data demonstrate directly that the 66 kDa protein is derived from GP IIIa and contains the PlAl alloantigen.

Antigens, Human Platelet↗

Immunocytochemical distribution of extracellular matrix receptors in human osteoclasts: a beta 3 integrin is colocalized with vinculin and talin in the podosomes of osteoclastoma giant cells.

Human osteoclasts (OCLs) obtained from cell suspensions of surgically excised giant cell bone tumors (osteoclastomas) were attached to glass coverslips and analyzed by immunofluorescence with antibodies to integrins and cytoskeletal proteins. It was found that in OCLs (i) podosomes, identified by their F-actin core and by interference reflection microscopy, were predominantly found in a peripheral belt as described in avian OCLs; (ii) each F-actin core was surrounded by a ring of vinculin and talin; (iii) beta 1 integrin was diffuse in the ventral membrane; (iv) beta 3 integrin was distributed in intensely fluorescent rings surrounding F-actin cores; (v) beta 2 integrin was absent; (vi) beta 4 integrin was absent. The macrophages detected in the same coverslips displayed podosomes containing beta 2 but not beta 3, fibroblasts showed adhesion plaques positive for beta 1 and beta 3 but not for beta 2, and platelets were intensely positive for beta 3. These results indicate that OCLs produce an integrin complex that is absent in the monocyte-macrophage lineage.

Actins↗

Interleukin-1beta expression is induced by adherence and is enhanced by Fc-receptor binding to immune complex in THP-1 cells.

Adherence of monocytes to endothelial cells and subsequently to basement membrane represents the initial steps in monocyte migration from the vasculature to the interstitium. We investigated the role of adhesion to endothelial cells and basement membrane in the induction of the cytokine IL-1beta. We demonstrated that mRNA for IL-1beta is induced in adherent THP-1 cells, but not in a matrix-specific manner. Adherence to fibrinogen, however, causes an increase in mRNA for IL-1beta. A background level of IL-1beta mRNA induction was observed in cells adherent to all matrices, including the non-specific human serum albumin substrate, as compared to non-adherent cells cultured in teflon troughs. In addition, antibodies to CD11a, CD11b, beta1 integrin, VLA4, (alpha)v(beta)3 (VNR), and ICAM-1 did not induce significant IL-1beta mRNA when THP-1 cells were adherent to those immunoglobulins. THP-1 cells adherent to immune complexes of anti-CD11a, anti-CD11b, anti-VLA4, anti-VNR, and anti-ICAM-1 showed greater mRNA induction than cells adherent to primary antibodies alone. THP-1 cells adherent to non-specific immune complexes gave the highest level of mRNA induction. Secretion of IL-1beta protein, measured by ELISA at 24 h, was greatest when cells were adherent to immobilized immune complexes or to fibrinogen. Our results demonstrate that a general adherence-induced increase in IL-1beta gene expression is greatly enhanced by the presence of immune complex.

Antigen-Antibody Complex↗

Integrin activation and cytoskeletal interaction are essential for the assembly of a fibronectin matrix.

Fibronectin (Fn) matrices are vital to vertebrate development and wound healing and modulate tumorigenesis. We used a recombinant Fn-binding integrin alpha IIb beta3, to define rules for integrin-initiated Fn matrix formation. We report the following. First, multiple Fn-binding integrins can support matrix assembly; their activation state controls fibrillogenesis. Second, Fn binding to cells expressing an activated integrin is necessary but not sufficient for matrix assembly. Additional "postoccupancy" events involving the integrin beta, but not the alpha subunit, cytoplasmic domain are needed. Third, these postoccupancy events require an intact actin cytoskeleton. We propose a model for integrin involvement in Fn fibrillogenesis that reconciles previous paradoxes and suggests novel approaches to the therapeutic control of Fn matrix assembly.

Actins↗

Quantitative determination of platelet surface alloantigens using a monoclonal probe.

A monoclonal antibody with specificity for the Fc portion of IgG was used to determine the number of IgG alloantibody molecules bound at saturation to alloantigens of the PlA1 and HLA systems on normal human platelets. In preliminary studies, it was found that the number of cell-bound IgG molecules recognized by this probe correlates well with the number measured by electroimmunoassay, an independent measure of alloantibody binding. PlA1-positive platelets could be divided into two groups binding 34,000-43,000 or 19,000-24,000 alloantibody molecules. Family studies and studies with a cytolytic assay showed that the former group is homozygous and the latter heterozygous for PlA1. Because the number of glycoprotein IIIa (GPIIIa) molecules carrying the PlA1 determinant on the surface of normal platelets is thought to be about 40,000, these findings suggest that each GPIIIa molecule carries one PlA1 determinant. The number of class I HLA molecules expressed on normal platelets was considerably smaller than the number of PlA1 determinants, ranging from 4400 to 10,000 (HLA-A2), 870 to 8400 (Bw4), and 1300 to 5800 (Bw6). Preliminary analysis indicates that stronger or weaker expression of Bw4 and of Bw6 correlates with certain "private" HLA-B determinants carried on the HLA-B molecule as found in previous studies using an indirect method to measure alloantigen density. These findings appear to explain why antibodies reactive with platelet-specific antigens such as PlA1 react more strongly with platelets than HLA-specific antibodies in most serologic tests. The weak expression of HLA determinants on platelets of some subjects may account for the less than perfect correlation between in vitro compatibility tests and post-transfusion platelet survivals observed in most studies.

Antibodies, Monoclonal↗