Platelet factor XI is associated with the platelet plasma membrane and has an apparent M r (220,000 nonreduced, 55,000 reduced) different from that of plasma factor XI. However, the site of synthesis and the nature of platelet factor XI are not known. Using reverse transcriptase polymerase chain reaction, 12 out of 13 exons (all except exon V) coding for mature plasma factor XI were amplified from human platelet mRNA. The sequence of each of these exons was identical to that of plasma factor XI. In situ amplification and hybridization of factor XI mRNA was positive for exon III and negative for exon V in platelets and negative for both exons in other blood cells. By Northern hybridization, a factor XI mRNA transcript of ϳ1.9 kilobases was detected in megakaryocytic cells, and one of ϳ2.1 kilobases was detected in liver cells. Factor XI cDNA was cloned from a megakaryocyte library and sequenced. Exon V was absent, and the splicing of exon IV to exon VI maintained the open reading frame without alteration of the amino acid sequence except for the deletion of amino acids Ala 91 -Arg 144 within the amino-terminal portion of the Apple 2 domain. Thus, platelet factor XI is an alternative splicing product of the factor XI gene, localized to platelets and megakaryocytes but absent from other blood cells.Plasma coagulation factor XI is a glycoprotein present in human plasma at a concentration of ϳ30 nM as a zymogen that, when converted by limited proteolysis to an active serine protease, participates in the contact phase of blood coagulation. This zymogen is an unique plasma coagulation enzyme because it exists as a homodimer (M r ϳ143,000) consisting of two identical polypeptide chains linked by disulfide bonds (1, 2).The sequence of the human factor XI gene has been elucidated by sequencing of the cDNA inserts coding for factor XI from two different phage genomic libraries (3,4 Factor XI coagulant activity and antigen are present in wellwashed platelet suspensions, and the activity accounts for about 0.5% of the total factor XI activity in blood (5, 6). About half of factor XI-deficient patients have defective hemostasis, whereas the remainder do not experience abnormal bleeding even in the absence of plasma factor XI (7). Subcellular fractionation studies have shown that factor XI activity is enriched in the platelet membrane fraction (8). The washed platelets and isolated platelet membranes obtained from a factor XIdeficient donor without a history of excessive bleeding had normal quantities of factor XI-like activity and normal behavior in the contact phase of coagulation (9). Thus, the functional significance of factor XI associated with platelets is not clear, but it may play a role in maintaining normal hemostasis, possibly complementing plasma factor XI deficiency (9).Previous studies employing immunofluorescence, immunoelectrophoresis, and immunoprecipitation utilizing monospecific polyclonal anti-factor XI antibodies have demonstrated the presence in and partial purification from human platelets of a molecule ...

Activated factor VIII (FVIIIa) forms a procoagulant complex with factor IXa on negatively charged membranes, including activated platelet surfaces. Membrane attachment involves the FVIII C2 domain; involvement of the adjacent C1 domain has not been established. Binding of recombinant FVIII C1C2 and C2 proteins to platelets was detected by flow cytometry using (1) anti-C2 monoclonal antibody ESH8 followed by a phycoerythrinlabeled secondary antibody; (2) biotinylated C1C2 detected by phycoerythrinlabeled streptavidin, and (3) C1C2 and C2 site-specifically labeled with fluorescein. Highest binding and lowest background were obtained using fluoresceinconjugated proteins. More than 90% of activated platelets bound C1C2, compared with approximately 50% for equimolar C2. Estimates using fluorescent microbeads indicated approximately 7000 C1C2-binding sites per platelet, approximately 1400 for C2, and approximately 3000 for fluorescein-labeled FVIIIa. Unlike C2 or FVIII(a), C1C2 bound to approximately 700 sites/platelet before activation. C1C2 binding to activated platelets appeared independent of von Willebrand factor and was competed effectively by FVIII(a), but only partially by excess C2. Fluorescein-labeled FVIIIa was competed much more effectively by C1C2 than C2 for binding to activated platelets. Two monoclonal antibodies that inhibit C2 binding to membranes competed platelet binding of C2 more effectively than C1C2. Thus, the C1 domain of FVIII contributes to platelet-binding affinity. IntroductionFactor VIII (FVIII) circulates in plasma in a noncovalent complex with von Willebrand factor (VWF); this interaction is mediated by the FVIII C2 domain and an acidic sequence prior to the A3 domain. [1][2][3] Upon proteolytic activation, FVIIIa is released from VWF as a heterotrimer composed of the A1 and A2 domains plus the FVIIIa light chain, A3-C1-C2. Activated platelet membranes expose negatively charged phosphatidylserine (PS), which increases from 2% to 10% or more of the surface phospholipids upon activation. 4,5 FVIIIa forms a complex with FIXa and calcium on negatively charged phospholipid membranes, enhancing FIXa catalysis 100 000-to 200 000-fold. [6][7][8] Although FVIII can bind to FIXa on phospholipids, 9 or directly to activated platelets, 10 FVIIIa is required for procoagulant activity. 9 A hydrophobic surface on the FVIII(a) C2 domain 11 becomes buried in the phospholipid membrane upon binding, 12,13 and basic C2 residues make favorable charge-charge interactions with negatively charged PS head groups. Although FVIIIa and the light chain bind to PS-containing vesicles and activated platelets with similar affinities, the affinity of the recombinant C2 domain is 5-to 100-fold lower, 10,14,15 suggesting possible roles for the C1 and/or A3 domains.To address the potential role of the C1 domain in FVIII(a) attachment to platelets, a recombinant human FVIII C1C2 protein (residues 2020-2332) was produced in Escherichia coli, refolded, and characterized. The binding of C1C2 to platelet surfaces both before ...

Summary. Epitopes recognized by factor VIII (FVIII) inhibitors of Chinese origin were analysed by immunoblotting with full-length recombinant FVIII (rFVIII), thrombinactivated FVIII (FVIIIa) and 16 FVIII fusion proteins synthesized by bacteria. Twenty-eight patients, 12 with haemophilia A and 16 with autoimmune diseases, were recruited. Antibodies from 22 patients showed reactivity with rFVIII, 20 with FVIIIa, and one reacted only with FVIII fusion proteins. Of these 22 cases, most were reactive with A2-a2 and A3-C1-C2 of FVIII(a). Of the nine cases that depicted binding to the fusion proteins, three were reactive with the A domains, three with only the B domain, and the other three with both the A and B (or C) domains. An epitope for a neutralizing antibody of a haemophilia A patient, designated TWN-112, was localized to residues 323±390, specified by FVIII fusion proteins. The same epitope also appeared on an FVIII-expression phage library screening. Immunoabsorption of antibodies from with the epitope reduced the neutralizing activity of the inhibitor by 33%. The incidence of a1 of FVIII is higher, and that of a3 is lower, than previously reported. Two novel epitopes, reported for the first time in this paper, were localized on the 8B2 (amino acid residues 1022±1204) and 8A2(V) (residues 673±740) fusion proteins. These two epitopes were able to reduce inhibitory antibody activity by 24% and 25% respectively. Changes of FVIII fragment specificity were also observed in one of six patients for whom multiple samples, collected at different times, were available. Our initial finding showed that the FVIII inhibitors in these Chinese patients shared epitopes with those of patients from very different genetic backgrounds, suggesting a common mechanism for the development of FVIII inhibitors.

The aquatic fern Marsilea quadrifolia produces different types of leaves in response to changes in natural environment and culture conditions. When the conditions are in favor of producing the submerged-type leaves, exogenous application of the plant hormone abscisic acid (ABA) induces the formation of aerial-type leaves. Tissues responsive to ABA were localized to the shoot apical meristem and the associated organ primordia. From these tissues, at least two tiers of ABA-regulated early genes were identified, including seven primary genes and seventeen secondary genes. These genes, designated ABRH for ABA-responsive heterophylly, showed diverse expression patterns during the course of heterophyllous induction. Changes in the transcript level of ABRH genes started early, within 0.5-1.0 h after the addition of ABA to the culture medium. Some changes were transient while the others were persistent. The ABRHs contain extensive sequence homology to known genes, including those encoding transcription factors, protein kinases, membrane transporters, metabolic enzymes, structural proteins and those encoded by the chloroplast genome. Identification of these ABRHs is a first step toward the understanding of the regulation mechanisms of heterophylly, and the results suggest the involvement of novel metabolic and regulatory pathways in ABA-controlled morphogenesis.