We measured reticulated platelets (RPs) and plasma glycocalicin (GC) and thrombopoietin (TPO) levels simultaneously in 107 thrombocytopenic patients to clarify the diagnostic value of these tests for discriminating hyperdestructive from hypoplastic thrombocytopenia. The percentage of RPs and GC index (plasma GC level normalized for the individual platelet count) were markedly elevated in patients with idiopathic thrombocytopenic purpura (ITP) but normal or slightly elevated in patients with aplastic anemia (AA) or chemotherapy-induced thrombocytopenia (ChemoT). For RP percentage for diagnosing hyperdestructive thrombocytopenia the sensitivity and specificity were excellent but were lower for the GC index. Absolute RP counts and plasma GC levels were markedly decreased and plasma TPO levels markedly elevated in patients with AA or ChemoT, but absolute RP counts and plasma GC levels were moderately decreased and plasma TPO levels only slightly elevated in patients with ITP. The sensitivity and specificity of plasma TPO levels for diagnosing hypoplastic thrombocytopenia were excellent. Using the RP percentage and plasma TPO levels in combination improved specificities. Simultaneous measurement of RP percentage and plasma TPO level may help discriminate thrombocytopenia of unknown cause in routine hematologic practice.

To elucidate genetic abnormalities in type I CD36 deficiency, we analyzed 28 Japanese subjects whose platelets and monocytes/macrophages lacked CD36 on their surface. We identified two novel mutations in the CD36 gene. One was a complex deletion/insertion mutation, in which 3 bp, GAG, were deleted at nucleotide (nt) 839-841, and 5 bp, AAAAC, were inserted at the same position (839-841del-->insAAAAC). Mutation 839-841del-->insAAAAC led to a frameshift and appearance of a premature stop codon; it was also accompanied with a marked reduction in the amount of CD36 mRNA. The other was a 12-bp deletion at nt 1438-1449 (1438-1449del) accompanied with or without skipping of exon 9 (nt 959-1028). Mutation 1438-1449del led to an inframe 4-amino-acid deletion, whereas exon 9 skipping led to a frameshift and the appearance of a premature stop codon. Expression assay revealed that both 1438-1449del and exon 9 skipping directly caused impairment of the surface expression of CD36. A survey of the five known mutations including 839-841del-->insAAAAC and 1438-1449del in type I CD36-deficient subjects demonstrated that the five mutations covered more than 90% of genetic defects among them and that the substitution of T for C at nt 478 (478C-->T) was the most common mutation with more than 50% frequency. However, none of the four subjects that possessed isoantibodies against CD36 had 478C-->T, suggesting that 478C-->T prevents the production of isoantibodies against CD36.

Integrin ␣ v ␤ 3 has been implicated in angiogenesis and other biological processes. However, the ligand-binding sites in ␣ v , a non-I-domain ␣ subunit, remain to be identified. Recently in ␣ IIb , the other partner of the ␤ 3 subunit, several discontinuous residues important for ligand binding were identified in the predicted loops between repeats 2 and 3 (W3 4-1 loop) and within repeat 3 (W3 2-3 loop). Based on these findings, alaninescanning mutagenesis in 293 cells was used to investigate the role of these loops (cys- teine IntroductionIntegrins are a large family of ␣␤ heterodimeric adhesion receptors that is often subdivided into groups based on 8 known integrin ␤ subunits. 1 The ␤ 3 -integrin subfamily is composed of ␣ v ␤ 3 , originally identified as the vitronectin receptor, and ␣ IIb ␤ 3 , a plateletspecific receptor for fibrinogen and von Willebrand factor. ␣ IIb ␤ 3 plays a crucial role in platelet aggregation, normal hemostasis, and pathological thrombus formation. 2 On the other hand, ␣ v ␤ 3 is expressed in a number of tissues, including platelets, endothelial cells, vascular smooth muscle cells, and osteoclasts, and it plays a key role in angiogenesis and bone resorption. 3,4 The ␣ IIb and ␣ v subunits are homologous and 36% identical in primary amino acid sequence, 5 and most ligands that bind to ␣ IIb ␤ 3 , including fibrinogen, von Willebrand factor, and vitronectin, also bind to ␣ v ␤ 3 . However, there are some distinctive features between these 2 integrins. 3 First, the ␣ IIb subunit has been found only in combination with ␤ 3 , whereas ␣ v can associate with at least 5 ␤ subunits (␤ 1 , ␤ 3 , ␤ 5 , ␤ 6 , and ␤ 8 ). 6 Second, some ligands, such as osteopontin, matrix metalloproteinase-2, and adenovirus penton base, bind to ␣ v ␤ 3 but not to ␣ IIb ␤ 3 . Third, treatment of ␣ IIb ␤ 3 with ethylenediamine tetraacetic acid (EDTA) at 37°C dissociates the complex into its individual subunits; ␣ v ␤ 3 remains a heterodimer. Finally, the ligand-binding function of ␣ v ␤ 3 , but not ␣ IIb ␤ 3 , is suppressed by calcium (Ca 2ϩ ). 7 Generally, all integrins require divalent cations for ligand recognition, and multiple residues important for ligand binding have been identified on both ␣ and ␤ subunits. 8 The N-terminal region of integrin ␣ subunits has 7 repeats of homologous sequences of about 60 amino acid residues. Some integrin ␣ subunits (eg, ␣ 2 , ␣ L , and ␣ M ) contain an inserted domain of about 200 amino acids residues (the I-domain) between the second and the third repeats in the ␣ subunit, which is critically involved in ligand binding. 9,10 The crystal structure of the I-domain has been determined, and the metal ion-dependent adhesion site (MIDAS) motif that contributes to cation binding, as well as ligand binding, has been clarified. 11 Interestingly, a MIDAS-like motif essential for the ligand-binding function was also identified in integrin ␤ subunits. 12,13 On the other hand, integrin ␣ subunits, such as ␣ v , ␣ IIb , and ␣ 4 , do not have the I-domain. The structural basis for the i...

The molecular basis for the interaction between a prototypic non-I-domain integrin, ␣ IIb ␤ 3 , and its ligands remains to be determined. In this study, we have characterized a novel missense mutation (Tyr143His) in ␣ IIb associated with a variant of Glanzmann thrombasthenia. Osaka-12 platelets expressed a substantial amount of ␣ IIb ␤ 3 (36%-41% of control) but failed to bind soluble ligands, including a high-affinity ␣ IIb ␤ 3 -specific peptidomimetic antagonist. Sequence analysis revealed that Osaka-12 is a compound heterozygote for a single 521

Localization of epitopes for plateletassociated (PA) anti-GPIIb-IIIa (␣ IIb ␤ 3 ) autoantibodies in chronic immune thrombocytopenic purpura remains elusive. Previous studies suggest that PA antibodies recognize the tertiary structure of intact glycoprotein (GP) IIb-IIIa. To localize their epitopes using antigen-capture enzymelinked immunosorbent assay (ELISA), the reactivity of 34 PA anti-GPIIb-IIIa antibodies was examined with recombinant GPIIbIIIa having a defect in ligand-binding sites in either GPIIb or GPIIIa, and no major conformational change was induced: KO variant GPIIb-IIIa was attributed to a 2-amino acid insertion between residues 160 and 161 in the W3 4-1 loop in GPIIb, and CAM variant GPIIb-IIIa was attributed to D119Y in GPIIIa. In one third (11 of 34) of the patients, PA antibodies showed a marked decrease (less than 50%) in reactivity with KO compared with wild-type GPIIb-IIIa. Their reactivity was also impaired against GPIIbD163A-IIIa. In sharp contrast, they reacted normally with CAM GPIIb-IIIa. OP-G2, a ligand-mimetic monoclonal antibody, markedly inhibited their binding to GPIIb-IIIa in patients with im- IntroductionChronic immune thrombocytopenic purpura (ITP) is an autoimmune disorder characterized by the early destruction of platelets from antiplatelet autoantibodies. [1][2][3] Autoantibodies from most patients with ITP are mainly directed to the platelet membrane glycoprotein (GP) IIb-IIIa (integrin ␣ IIb ␤ 3 ) or GPIb-IX. 4,5 It has been demonstrated that platelet-associated (PA) autoantibodies, rather than serum antibodies, play a key role in platelet destruction. 6,7 Although a few studies demonstrate the localization of autoantigens on GPIIb or GPIIIa for serum antibodies, [8][9][10][11] characterization of the antigenic epitope(s) for PA autoantibodies remains elusive. 12-14 Dissociation of the GPIIb-IIIa complex into free GPIIb and GPIIIa by EDTA treatment markedly impaired the reactivity of PA anti-GPIIb-IIIa autoantibodies, suggesting that most PA autoantibodies recognize cation-dependent conformation(s) of GPIIbIIIa. 15,16 Characterization by using large recombinant GPIIIa peptides failed to localize the autoantigenic epitopes on GPIIb-IIIa, 17 and our recent study indicated that PA anti-GPIIb-IIIa (␣ IIb ␤ 3 ) autoantibodies do not react with ␣ V ␤ 3 . 18 These findings suggest that PA autoantibodies are highly GPIIb-IIIa specific and that they recognize the tertiary structure of intact GPIIb-IIIa. In other words, it is likely that most autoantigenic epitopes are localized in either the GPIIb-IIIa complex or the cation-dependent conformations on GPIIb or GPIIIa.The GPIIb-IIIa complex (␣ IIb ␤ 3 ), a noncovalently associated, divalent cation-dependent heterodimer, is a prototypic integrin and plays a crucial role in normal hemostasis and platelet aggregation as a physiologic receptor for fibrinogen and von Willebrand factor. 19,20 The interaction of these ligands with GPIIb-IIIa is mediated at least in part by an RGD sequence. Glanzmann thrombasthenia (GT) is a rare autosomal r...