ALBERT

Description: Integrin α/β heterodimer adopts a compact bent conformation in the resting state, and upon activation undergoes a large-scale conformational rearrangement. During the inside-out activation, signals impinging on the cytoplasmic tail of β subunit induce the α/β separation at the transmembrane and cytoplasmic domains, leading to the extended conformation of the ectodomain with the separated leg and the opening headpiece that is required for the high-affinity ligand binding. It remains enigmatic which integrin subunit drives the bent-to-extended conformational rearrangement in the inside-out activation. The β3 integrins, including αIIbβ3 and αVβ3, are the prototypes for understanding integrin structural regulation. The Leu33Pro polymorphism located at the β3 PSI domain defines the human platelet-specific alloantigen (HPA) 1a/b, which provokes the alloimmune response leading to clinically important bleeding disorders. Some, but not all, anti–HPA-1a alloantibodies can distinguish the αIIbβ3 from αVβ3 and affect their functions with unknown mechanisms. Here we designed a single-chain β3 subunit that mimics a separation of α/β heterodimer on inside-out activation. Our crystallographic and functional studies show that the single-chain β3 integrin folds into a bent conformation in solution but spontaneously extends on the cell surface. This demonstrates that the β3 subunit autonomously drives the membrane-dependent conformational rearrangement during integrin activation. Using the single-chain β3 integrin, we identified the conformation-dependent property of anti–HPA-1a alloantibodies, which enables them to differently recognize the β3 in the bent state vs. the extended state and in the complex with αIIb vs. αV. This study provides deeper understandings of integrin conformational activation on the cell surface.

Description: Key Points The genome of iPSCs has been edited to encode antigenically-distinct human platelet alloantigens. The iPSC-derived megakaryocyte progenitor cells express the designed alloantigens for diagnostic, investigative, and future therapeutic use.

Description: Heparin induced thrombocytopenia and thrombosis (HIT) is a major cause of morbidity and mortality in patients given heparin and accurate and timely diagnosis is critical for successful management of this condition. HIT is caused by antibodies specific for a basic platelet protein, PF4, in a complex with heparin. An assay widely used to detect such antibodies, the PF4-based ELISA, is highly sensitive for antibody detection but not specific for the clinical disorder, HIT. An alternative test, the serotonin release assay (SRA) is quite specific for the clinical disorder, but is demanding technically and is performed routinely only by a few laboratories. Assigning a HIT diagnosis wrongly (due to lack of diagnostic specificity in the ELISA) can lead to treatment with alternative anticoagulants which are expensive, carry higher bleeding risks, and (unlike heparin) lack a specific antidote. Here, we describe a newly developed assay that has operating characteristics similar to the SRA in that it preferentially recognizes “activating” antibodies likely to be pathogenic but is much less demanding technically. The assay end point for platelet activation is FcγRIIa-mediated expression of p-selectin (CD62p) triggered by binding of antibody to PF4 in a complex with platelet surface glycosaminoglycans, a process that mimics the mechanism by which HIT antibodies are thought to activate platelets and predispose to thrombosis in vivo. The unique feature of the assay is addition of exogenous PF4 to platelets, an approach not previously used in diagnostic testing for HIT (Fig. 1). We compared test performance using a set of blinded HIT samples that were either SRA positive or SRA negative, but reacted equally well in the PF4 ELISA. The new assay correctly identified “pathogenic” HIT antibodies (i.e. those positive in the SRA) in 36 of 41 patient samples using a threshold of 〉10% increase in p-selectin expression relative to pooled negative serum (Fig 2). Five samples were originally “misclassified”, with the p-selectin expression assay having an area under the curve (AUC, a measure of diagnostic accuracy) of 0.90. Four samples were “false positives” i.e., were SRA negative but positive in the p-selectin assay (Fig 2, Red and blue circles) and one was “false negative” (Fig 2, Green circle). Three of the four “false positives” (red circle) reacted strongly in the p-selectin assay, were inhibited with high dose heparin and failed to react in the absence of added PF4 (data not shown). These findings strongly suggested the presence of a heparin-depending antibody in these 3 samples, and repeat SRA demonstrated that all three were actually SRA+. Thus, 39 of 41 HIT samples produced test results concordant with SRA, with an AUC for the new assay of 0.98 (1.0 indicates “perfect” performance). We postulate that the ability of the p-selectin expression assay to preferentially identify HIT antibodies that activate platelets is a consequence of the close similarity between the mechanics of the test and the pathogenic process that takes place in vivo in a patient with HIT. Testing of a larger prospective series of HIT samples to further assess diagnostic utility of the assay is in progress. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3506 Poster Board III-443 A diagnosis of heparin-induced thrombocytopenia (HIT) is confirmed with support from the laboratory. Antibodies associated with HIT are specific for complexes made up of heparin and platelet factor 4 (PF4) and can be detected in a solid phase ELISA (PF4 ELISA). However, a functional test, the serotonin release assay (SRA) is regarded by many to be the “gold standard” for laboratory investigation of this disorder. In our facility, the SRA is performed by incubating serotonin-labeled platelets (pooled from three different group O donors) with test serum and low dose (0.1 units/ml) or high dose (100 units/ml) heparin and determining the percentage of total serotonin released. Release of serotonin (20-100%) with low dose heparin and inhibition of this release with high dose heparin is considered to be “positive” for platelet-activating HIT antibodies. Sera from some patients cause serotonin release with low dose heparin that is not inhibited with high dose heparin. The significance for these “indeterminate” reactions is unclear, but they are considered not to reflect the presence of “true” HIT antibodies. We studied selected serum samples from 238 patients referred for HIT testing. Of these, 119 tested “true” positive and 117 produced “indeterminate” reactions in the SRA. The same samples were tested for the presence of antibodies reactive with beads coated with various Class I HLA antigens using a flow cytometric bead assay (Flow PRA, One Lambda). Sera producing at least 30% release in SRA and reactive with at least 20% of the bead panel were selected for analysis. As shown in Figure 1, there was a high correlation between the likelihood of an “indeterminate” SRA test result and the presence of Class I HLA antibodies (p /=20% No. with PRA /=30% Release and Uninhibited by High Dose Heparin) 91 19 110 SRA “True Positive” (〉/=30% Release and Inhibited by High Dose Heparin) 36 72 108 As expected, there was a significant correlation between the strength of reactions produced by individual “true positive” sera in the SRA (% release) and in the PF4/heparin ELISA (O.D. value). However, in analyzing 38 sera with “true positive” test results in the SRA, we identified two that were negative in the PF4 ELISA and contained broad Class I HLA reactivity (reactive with 100% and 41% of the panel, respectively). We conclude 1) Class I HLA antibodies are the major cause of “indeterminate” reactions in the serotonin release assay and 2) A subset of these antibodies can be inhibited by high dose heparin and therefore mimics the behavior of “true” HIT antibodies in the SRA. Unless the PF4 ELISA test is used together with the SRA, this type of reaction could lead to an erroneous diagnosis of HIT. Disclosures: No relevant conflicts of interest to declare.

Description: Drug-induced immune thrombocytopenia (DITP) is often suspected in patients with acute thrombocytopenia unexplained by other causes, but documenting that a drug is the cause of thrombocytopenia can be challenging. To provide a resource for diagnosis of DITP and for drug safety surveillance, we analyzed 3 distinct methods for identifying drugs that may cause thrombocytopenia. (1) Published case reports of DITP have described 253 drugs suspected of causing thrombocytopenia; using defined clinical criteria, 87 (34%) were identified with evidence that the drug caused thrombocytopenia. (2) Serum samples from patients with suspected DITP were tested for 202 drugs; drug-dependent, platelet-reactive antibodies were identified for 67 drugs (33%). (3) The Food and Drug Administration's Adverse Event Reporting System database was searched for drugs associated with thrombocytopenia by use of data mining algorithms; 1444 drugs had at least 1 report associated with thrombocytopenia, and 573 (40%) drugs demonstrated a statistically distinctive reporting association with thrombocytopenia. Among 1468 drugs suspected of causing thrombocytopenia, 102 were evaluated by all 3 methods, and 23 of these 102 drugs had evidence for an association with thrombocytopenia by all 3 methods. Multiple methods, each with a distinct perspective, can contribute to the identification of drugs that can cause thrombocytopenia.

Description: Acute thrombocytopenia is one of the most common serious adverse reactions to drugs and can be caused by many drugs. For evaluation of unexpected thrombocytopenia, it is important to know the relative risks of thrombocytopenia among the patient’s current medications. However identification of drugs that can cause thrombocytopenia is not standardized; multiple distinct methods are used: detection of drug-dependent antibodies, analysis of published case reports to establish the level of evidence for the drug as a cause of thrombocytopenia (http://moon.ouhsc.edu/jgeorge), and reporting to MedWatch, the FDA AERS. We have previously compared drugs that had “definite” or “probable” evidence for causing thrombocytopenia in published reports to drugs that had a significant association with thrombocytopenia determined by data mining of the FDA AERS database, defined as a signal of disproportionate reporting (SDR) that exceeded a standard predetermined value (Li, et al. Blood2006;108:140a). We have now expanded our analysis to include flow cytometry detection of DDab in the serum of patients with suspected drug-induced thrombocytopenia. 401 drugs have been suspected as a cause of thrombocytopenia by serum samples submitted for identification of DDab and in case reports. All 401 drugs, including drugs that were and were not shown to be associated with DDab and also drugs that did or did not have “definite” or “probable” evidence for causing thrombocytopenia in case reports, were searched for in the AERS database using a data mining algorithm to identify a significant association with thrombocytopenia. In this analysis, drugs that were not reported in a publication, were not tested for DDab, or were not found in the AERS database were coded as not significant for that method. 204 (51%) of the 401 drugs were significantly associated with thrombocytopenia by 1 or more methods; DDab identified 12% (47), case reports 19% (75), and data mining 36% (143). However, there was limited agreement among these 3 methods for identifying a significant association with thrombocytopenia. Significant by all 3 methods 13 drugs (3%) Significant by any 2 methods 35 drugs (9%) Significant only by detection of DDab 21 drugs (5%) Significant only by case reports 39 drugs (10%) Significant only by data mining 96 drugs (24%) Not significant by any of the 3 methods 197 drugs (49%) None of the 3 methods are sufficient to identify all drugs capable of causing thrombocytopenia. Data mining is a screening tool of existing data and therefore may be more sensitive but less specific than demonstration of DDab and reported clinical evidence. Reports to MedWatch are simple to submit but the reliability is uncertain. Critical assessment of clinical evidence from published case reports may be more specific for identifying drugs that can cause thrombocytopenia, but substantial effort is required to publish a case report. Detection of DDab specifically identifies drugs that can cause thrombocytopenia and also provides understanding of the biologic mechanisms, but tests for DDab are not standardized in routine clinical laboratories. Conclusions. Use of multiple approaches is important to enhance post-marketing surveillance and to provide a comprehensive understanding of drug-induced thrombocytopenia.

Description: The molecular basis of the HNA-3a/b (5b/a) leukocyte antigen system has not yet been defined despite evidence that HNA-3a–specific antibodies are particularly prone to cause severe, often fatal, transfusion-related lung injury. We used genome-wide single nucleotide polymorphism scanning and sequencing of DNA from persons of different HNA-3a/b phenotypes to identify a single single nucleotide polymorphism in exon 7 of the CLT2 gene (SLC44A2) that predicts an amino acid substitution in the first extracellular loop of choline transporter-like protein 2, a member of the choline transporter-like protein family of membrane glycoproteins, and correlates perfectly with HNA-3a/b phenotypes (R154 encodes HNA-3a; Q154 encodes HNA-3b). Mass spectrometric analysis of proteins immunoprecipitated from leukocytes by anti–HNA-3a provided direct evidence that anti–HNA-3a recognizes choline transporter-like protein 2. These findings will enable large-scale genotyping for HNA-3a/b to identify blood donors at risk to have HNA-3a–specific antibodies and should facilitate development of practical methods to detect such antibodies and prevent transfusion-related lung injury.