Factor VIII Procoagulant Protein Interacts with Phospholipid Vesicles Via its 80 kDa Light Chain

1988 ◽  
Vol 60 (03) ◽  
pp. 442-446 ◽  
Author(s):  
G Kemball-Cook ◽  
S J Edwards ◽  
K Sewerin ◽  
L O Anderson ◽  
T W Barrowcliffe
Keyword(s):  
Factor Viii ◽  
Binding Site ◽  
Light Chain ◽  
Binding Sites ◽  
Light Chains ◽  
Phospholipid Vesicles ◽  
Polyacrylamide Gels ◽  
Phospholipid Binding

SummaryIn a previous report, we detailed the fractionation of polyclonal human anti-Factor VIII :C into a component directed exclusively against the phospholipid-binding site on Factor VIII (PL-site antibody) and another directed at other sites (non-PL-site antibody). The location on the F.VIII molecule of its PL-binding site has now been studied by two different methods using this fractionated 125I-labelled anti-F.VIII: C Fab’.The first method was modified from that of Weinstein et al. (Proc Natl Acad Sci USA 1981; 78: 5137-41), involving electrophoresis of F.VIII peptide-125I-Fab‘ A/F.VIII immunocomplexes in SDS-polyacrylamide gels. PL-site antibody reacted with F.VIII peptides of apparent Mr approximately 80 kDa and sometimes 160 kDa in plasma and concentrate, but not with larger peptides. Non-PL-site antibody, however, reacted with a range of peptides of apparent Mr 90 kDa to 280 kDa. In addition, when purified F.VIII containing heavy and light chains (HC + LC), and isolated LC peptides were analysed, PL-site antibody bound to LC peptides whereas non-PL-site antibody did not.The second method used the antibody pools in immunoradiometric assays (IRMA’s) of purified F.VIII peptides. Both labels measured similar amounts of F.VIII: Ag in a sample of purified F.VIII containing both HC and LC; on assaying an HC preparation, however, PL-site label measured only 2% of F.VIII: Ag found by non-PL-site label, indicating that PL-binding sites are absent in HC preparations.These results indicate that F.VIII binds to PL via its 80 kDa light chain.

Thrombin-Stimulated Platelets Have Functional Binding Sites For Factor VIIIa That Are Distinct From Phosphatidylserine

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3582-3582
Author(s):  
Jialan Shi ◽  
Valerie A Novakovic ◽  
Steven Pipe ◽  
Shannon Meeks ◽  
John (Pete) S. Lollar ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Factor Viii ◽  
Binding Site ◽  
Binding Sites ◽  
Conflicts Of Interest ◽  
Specific Binding ◽  
Phospholipid Vesicles ◽  
C2 Domain ◽  
Phospholipid Binding ◽  
Fviii Activity

Abstract Background Factor VIII (fVIII) functions as a co-factor for factor IXa on the membranes of stimulated platelets. Binding sites for fVIII(a) are expressed at two levels; thrombin induces 3,000 – 20,000 sites/platelet while the combination of collagen and thrombin or A28137 induce >50,000 sites/platelet. Hypothesis We hypothesized that binding sites for fVIII(a) on thrombin-stimulated platelets, are distinct from phosphatidylserine (PS), while those on maximally stimulated platelets are predominantly PS-containing sites. Corollaries were 1) that epitopes on fVIII interact with the non-PS sites and 2) that a macromolecule or a macromolecule complex comprises the binding sites on thrombin-stimulated platelets. Methods Platelets were purified on a density gradient and binding of fluorescein-labeled fVIII (fVIII-fluor) was measured by flow cytometry using a Becton Dickinson LSR-Fortessa flow cytometer. Factor VIII activity was measured in a discontinuous factor Xase assay using extruded phospholipid vesicles of composition PS:PE:PC 4:20:76 or platelets as the membrane source. Oligomeric fibrin was immobilized by incubating thrombin, 1 u/ml, with fibrinogen, 10 µg/ml for 10 min without mixing prior to addition of 59D8-Superose beads. Binding of fVIII-4 Ala to platelets was measured in complex with Alexa-488 labeled mAb GMA-8021, against the A2 domain. Polyphosphate was size-fractionated and recombinant PPX-MBD produced as previously described. Results Lactadherin, a phosphatidyl-L-serine-binding protein, competed for 97% of factor VIII-fluorescein (fVIII-fluor) binding sites on A23187-stimulated platelets but only 30% of binding sites on thrombin-stimulated platelets. Unlabeled fVIII competed with fVIII-fluor for all binding sites. A fVIII C2 domain mutant, with no measurable phospholipid binding - M2199A/F2200A/L2251A/L2252A (fVIII-4Ala) bound to only 3,000 – 5,000 sites on platelets stimulated with A23187 but to a similar number on thrombin-stimulated platelets with a KDof 7 nM. These data indicate that non-PS sites are dominant on thrombin-stimulated platelets but that PS-containing sites comprise at least 95% of sites on A23187-stimulated platelets. We evaluated a panel of mAb’s against the fVIII-C2 domain for platelet-specific inhibition of binding and function. mAb’s ESH4 and I54, with overlapping epitopes, blocked binding of fVIII to thrombin-stimulated platelets but only decreased affinity for PS-containing membranes. In 1-stage and 2-stage commercial aPTT assays ESH4 inhibited 28-33% of fVIII activity. In contrast, ESH4 inhibited 80% of fVIII activity on thrombin-stimulated platelets. mAb’s ESH8 and G99, with partially overlapping epitopes, decreased the affinity of fVIII-fluor for thrombin-stimulated platelets approx. 70% but had no effect on phospholipid binding. ESH8 inhibited 58 ± 8% of fVIII activity on thrombin-stimulated platelets but did not decrease activity supported by phospholipid vesicles. Because oligomeric fibrin is required for expression of most fVIII binding sites on thrombin-stimulated platelets (Phillips et al 2004; JTH 2:1806) we hypothesized that oligomeric, platelet-bound fibrin is a constituent of fVIII binding sites. fVIII-fluor bound to fibrin monomers and oligomers immobilized on mAb 59D8-Superose, detected in solution by flow cytometry. Binding was enhanced by mixing polyphosphate (polyP) with fibrinogen prior to thrombin, with a maximum gain in affinity at 0.1 µM elemental phosphorous. The apparent affinity of fibrin-polyP for fVIII-fluor was 2-12 nM, based on competition studies with unlabeled fVIII. Like binding to platelets, specific binding of fVIII to fibrin-polyP was blocked by mAb’s ESH4, I54 and diminished by ESH8, and G99. Thrombin-stimulated platelets, but not resting platelets, exhibited bound polyP, as detected by PPX-MBP, specific for polyP. Thus, bound polyP is present on thrombin-stimulated platelets under conditions that lead to binding of oligomeric fibrin. Conclusions These data indicate that thrombin-stimulated platelets bind fVIII via a non-PS binding site and that the binding is mediated by epitopes that have greater functional importance on platelets than on phospholipid vesicles. Platelet-bound oligomeric fibrin with polyP is a candidate for the non-PS binding site. These findings have clinical relevance to detection of inhibitory antibodies against fVIII. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Clathrin assembly involves a light chain-binding region.

1987 ◽  
Vol 105 (5) ◽  
pp. 2011-2019 ◽  
Author(s):  
G S Blank ◽  
F M Brodsky
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Heavy Chain ◽  
Light Chain ◽  
Intermediate Filament ◽  
Binding Sites ◽  
Light Chains ◽  
Binding Region ◽  
Intermediate Filament Proteins ◽  
Interaction Site

Two regions on the clathrin heavy chain that are involved in triskelion interactions during assembly have been localized on the triskelion structure. These regions were previously identified with anti-heavy chain monoclonal antibodies X19 and X35, which disrupt clathrin assembly (Blank, G. S., and F. M. Brodsky, 1986, EMBO (Eur. Mol. Biol. Organ.) J., 5:2087-2095). Antibody-binding sites were determined based on their reactivity with truncated triskelions, and were mapped to an 8-kD region in the middle of the proximal portion of the triskelion arm (X19) and a 6-kD region at the triskelion elbow (X35). The elbow site implicated in triskelion assembly was also shown to be included within a heavy chain region involved in binding the light chains and to constitute part of the light chain-binding site. We postulate that this region of the heavy chain binds to the interaction site identified on the light chains that has homology to intermediate filament proteins (Brodsky, F. M., C. J. Galloway, G. S. Blank, A. P. Jackson, H.-F. Seow, K. Drickamer, and P. Parham, 1987, Nature (Lond.), 326:203-205). These findings suggest the existence of a heavy chain site, near the triskelion elbow, which is involved in both intramolecular and intermolecular interactions during clathrin assembly.

scholarly journals Affinity labelling of the binding site of rabbit antibody. Evidence for the involvement of the hypervariable regions of the heavy chain

1974 ◽  
Vol 139 (1) ◽  
pp. 135-149 ◽  
Author(s):  
Christopher E. Fisher ◽  
Elizabeth M. Press
Keyword(s):  
Binding Site ◽  
Heavy Chain ◽  
Binding Sites ◽  
Light Chains ◽  
Rabbit Antibody ◽  
Heavy Chains ◽  
Group 4 ◽  
Hypervariable Regions ◽  
Affinity Labelling ◽  
Antibody Complex

The binding sites of rabbit antibodies with affinity for the haptenic group 4-azido-2-nitrophenyl-lysine have been specifically labelled by photolysis of the hapten–antibody complex. The extent of covalent labelling was 0.5–0.9mol of hapten bound/mol of antibody and, by using an immunoadsorbent, antibody with 1.3mol of hapten/mol was obtained. The antibody was specifically labelled in the binding site and the ratio of labelling of heavy and light chains was in the range 3.3–5.0. The labelled heavy chains were cleaved by CNBr treatment and after reduction and alkylation of the intrachain bonds, were digested with trypsin. Evidence is presented that two regions of the heavy chain, positions 29–34 and 95–114, together contain about 80% of the label on the heavy chain; these two regions respectively include two of the hypervariable regions of rabbit heavy chain.

scholarly journals Some factor VIII inhibitor antibodies recognize a common epitope corresponding to C2 domain amino acids 2248 through 2312, which overlap a phospholipid-binding site

Blood ◽  
1995 ◽  
Vol 86 (5) ◽  
pp. 1811-1819 ◽  
Author(s):  
D Scandella ◽  
GE Gilbert ◽  
M Shima ◽  
H Nakai ◽  
C Eagleson ◽  
...  
Keyword(s):  
Amino Acids ◽  
Factor Viii ◽  
Binding Site ◽  
Light Chain ◽  
Intrinsic Factor ◽  
Factor Viii Inhibitor ◽  
C2 Domain ◽  
Amino Acid Residues ◽  
Fviii Inhibitor ◽  
Human Factor Viii

The finding that human factor VIII (fVIII) inhibitor antibodies with C2 domain epitopes interfere with the binding of fVIII to phosphatidylserine (PS) suggested that this is the mechanism by which they inactivate fVIII. We constructed a recombinant C2 domain polypeptide and demonstrated that it bound to all six human inhibitors with fVIII light chain specificity. Thus, some antibodies within the polyclonal anti-light chain population require only amino acids within C2 for binding. Recombinant C2 also partially or completely neutralized the inhibitor titer of these plasmas, demonstrating that anti-C2 antibodies inhibit fVIII activity. Immunoblotting of a series of C2 deletion polypeptides, expressed in Escherichia coli, with inhibitor plasmas showed that the epitopes for human inhibitors consist of a common core of amino acid residues 2248 through 2312 with differing extensions for individual inhibitors. The epitope of inhibitory monoclonal antibody (MoAb) ESH8 was localized to residues 2248 through 2285. Three human antibodies and anti-C2 MoAb NMC-VIII/5 bound to a synthetic peptide consisting of amino acids 2303 through 2332, a PS- binding site, but MoAb ESH8 did not. These antibodies also inhibited the binding of fVIII to synthetic phospholipid membranes of PS and phosphatidylcholine, confirming that the blocked epitopes contribute to membrane binding as well as binding to PS. In contrast, MoAb ESH8 did not inhibit binding. As the maximal function of activated fVIII in the intrinsic factor Xase complex requires its binding to a phospholipid membrane, we propose that fVIII inhibition by anti-C2 antibodies is related to the overlap of their epitopes with the PS-binding site. MoAb ESH8 did not inhibit fVIII binding to PS-containing membranes, suggesting the existence of a second mechanism of fVIII inhibition by anti-C2 antibodies.

THE PHOSPHOLIPID-BINDING SITE OF FACTOR VIII IS LOCATED ON THE 80 kD LIGHT CHAIN

1987 ◽  
Author(s):  
G Kemball-Cook ◽  
S J A Edwards ◽  
K Sewerin ◽  
L-O Andersson ◽  
T W Barrowcliffe
Keyword(s):  
Binding Site ◽  
Light Chain ◽  
Binding Sites ◽  
Immunoaffinity Chromatography ◽  
The Other ◽  
Immunological Methods ◽  
Electrophoretic Separation ◽  
Antigenic Sites ◽  
Reducing Conditions ◽  
Sds Page

The binding of Factoi. VIII (F.VIII) peptides to phospholipid (PL) vesicles has been studied by two different methods involving the use of fractionated anti-F.VIII:C I-Fab123’pre viously reported, i-Fab123’ was fractionated by immunoadsorptionwith F.VIII-PL complexes into two pools:one binding only to PL-binding sites on F.VIIIsAg (PL-site antibody), the other directed against other antigenic sites (non-PL-site antibody).The first technique used was a modification of the method of Weinstein et al. (Proc.Natl.Acad.Sci.USA, 78, 5137-5141, 1981), and involved incubation of the two anti-F.VIII pool swith F.VIII-containing samples, followed by electrophoretic separation of the complexes on the basis of size in non-denaturing SDS gels: this technique allows qualitative analysis of antibody reactive peptides in highly impure samples. Non-PL-site pool reacted with a range of peptides with MrMapparent Mr 90 kD up to 280 kD, a similar pattern to that of ’heavy chain’(HC) peptides of F.VIII seen on SDS-PAGE under reducing conditions; the PL-site antibody, however, reacted only with peptides at apparent Mrs of 80 kD and sometimes150 kD, but not with bands of higher Mr a pattern more consistent with binding to light chain (LC) peptides. Thesame patterns with the two labels were seen in both plasma and F.VIII concentrateThe second approach employed the two labels described above in direct immunoradiometric assays (IFMA’s) on purified human F.VIII peptides prepared by immunoaffinity chromatography and ion exchange on Mono Q gel. Both PL-site and non-PL-site labels measured similar amounts of F.VIII m a sample containing both HC and LC peptides; however, on assaying a sample containing purified HC peptides alone, PL-site antibody measured only 2% of F.VIII:Ag found by non-PL-site label, indicating that PL-binding sites present in samples containing both HC and LC are absent in HC alone.Results from both these immunological methods indicate that the 80 kD LC peptide of F.VIII carries the PL-binding site.

scholarly journals Properties of the non-specific calcium-binding sites of rabbit skeletal-muscle myosin

1980 ◽  
Vol 185 (1) ◽  
pp. 265-268 ◽  
Author(s):  
J Wikman-Coffelt
Keyword(s):  
Skeletal Muscle ◽  
Light Chain ◽  
Binding Sites ◽  
Calcium Binding ◽  
Light Chains ◽  
Binding Properties ◽  
Skeletal Muscle Myosin ◽  
Heavy Chains ◽  
Calcium Binding Sites ◽  
Muscle Myosin

The non-specific Ca2+-binding sites of skeletal-muscle myosin are located on the light chains; with the dissociation of light chains there is a corresponding decrease in the number of Ca2+-binding sites on light-chain-deficient myosin. The released light chains have a decreased binding affinity. Myosin heavy chains indirectly influence the Ca2+-binding properties of light chains by increasing the affinity of light chains for bivalent cations; this influence varies with pH. Because of light-chain dissociation at low Ca2+ and/or Mg2+ concentrations, anomalies may exist when analyses of non-specific Ca2+-binding properties of myosin are assessed by dialysis equilibrium.

An Essential Role of the Factor VIII Light Chain in Facilitating Heavy Chain Secretion.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4034-4034
Author(s):  
Lingxia Chen ◽  
Juan Li ◽  
Hui Lu ◽  
Haiyan Jiang ◽  
Rita Sarkar ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Hek293 Cells ◽  
Light Chains ◽  
Trans Splicing ◽  
In Cis

Abstract Blood coagulation Factor VIII (FVIII) is secreted as a heterodimer consisting of a heavy and light chain. Both in vitro and in vivo studies have demonstrated that these chains can be expressed independently. The expressed heavy and light chains can reassociate with recovery of biological activities. These observations have been particularly useful in a gene therapy setting since vector packaging capacity for adeno-associated virus (AAV) is a limiting factor. However, it has been demonstrated that the FVIII heavy chain is expressed ~10–100-fold less efficiently compared to the light chain when expressed independently. Previously the FVIII F309S mutation in the context of B-domainless FVIII (FVIII-BDD) and enhanced glycosylations within the B-domain have been shown to improve factor VIII expression and secretion. However, our in vitro studies indicate that these improvements in secretion were not retained when expressing the heavy chain alone with the same modifications. Other sequences, possibly in the light chain, may facilitate secretion. To investigate this further, we designed an intein trans-splicing strategy to control the addition of light chain to the heavy chain before secretion. Using HEK293 cells, we cotransfected seperate intein light chain and intein heavy chain plasmids and compared results to single plasmid transfected cells. 48 hours post-transfection, FVIII-specific ELISA results demonstrated that cotransfection of intein heavy chain and intein light chain had a significant influence on total heavy chain secretion compared to intein heavy chain expression alone. The co-transfected intein heavy chain and intein light chain were efficiently ligated together yielding a biologically active single chain FVIII derivative as demonstrated by clotting assays and Western blot analysis. Therefore, heavy chain secretion was directly enhanced by the attachment of the light chain to the C-terminus of the heavy chain. A similar phenomenon was not found when heavy and light chains were simply co-expressed in the same cell. It suggested that light chain functioned in cis. Hydrodynamic injection of plasmids with intein heavy chain and intein light chain into hemophilia A mice led to a much higher level of FVIII secretion. The amount of functional FVIII expression reached 3–6 units/ml at peak level. In the absence of intein light chain, FVIII heavy chain secretion was approximately 100 fold less efficient in vivo. To map the key elements of FVIII light in helping FVIII secretion, we made deletion variants in the light chain. These mutants had a dominant negative effect in reducing FVIII and FVIII heavy chain secretion while increasing the level of intracellular FVIII accumulation. Collectively our results are consistent with the conclusion that the FVIII light chain plays a critical role in facilitating heavy chain secretion in cis; probably through helping FVIII heavy chain maintain correct configuration and folding. The strategy to manipulate FVIII light chain addition through intein mediated trans-splicing reaction may also be explored for human gene therapy.

Intact Immunoglobulin or Fragments Thereof Can Be Detected in Urine in a Proportion of Patients with Multiple Myeloma and Are Associated with Reduced Survival At Presentation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1828-1828
Author(s):  
Heinz Ludwig ◽  
Philip Young ◽  
Dejan Milosavljevic ◽  
Niklas Zojer ◽  
Wolfgang Hübl ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Overall Survival ◽  
Binding Site ◽  
Light Chain ◽  
Free Light Chain ◽  
Light Chains ◽  
Glomerular Injury ◽  
Female Ratio ◽  
Serum Free ◽  
Serum Free Light Chain

Abstract Abstract 1828 Introduction: Intact immunoglobulin or fragments thereof (intact/fragmented Ig) can be found in the urine due to nephrotic injury or the preferential scavenging of albumin by the renal FcRn receptor leading to immunoglobulin catabolism. Until now the occurrence, frequency and clinical impact of this phenomenon has not been assessed in patients with multiple myeloma (MM). Here we determine the incidence of intact/fragmented Ig in urine and evaluate its prognostic relevance. Patients and Methods: 94 patients with MM, median age 70 years old (range 41–87) with a male / female ratio 28/66, ISS stage I (48), stage II (23), stage III (28), 69 IgG (43 IgGk/26 IgGl) and 25 IgA (15 IgAk/7 IgAl) were enrolled. Serum free light chain concentrations (sFLC) were measured using commercially available immunoassays (Freelite™, The Binding Site, Birmingham, UK) and compared to electrophoresis results (Hydrasys, Sebia, Paris, France). Overall survival was estimated by the product limiting method of Kaplan-Meyer and survival was compared by the log rank test. Results: Overall, sFLC ratios had a greater sensitivity than urine immunofixation (uIFE) for the detection of monoclonal light chains 86/94 vs. 46/94. In 13/46 (28%) uIFE positive patients intact immunoglobulins or significant fragments (intact/fragmented Ig) thereof were detected, 12 IgG, (12/69, 17%) and 1 IgA (1/25, 4%). Three of these patients had normal urine protein concentrations (<250mg/L) and 2/13 patients had glomerular injury identified by increased levels of albumin excretion. There was no difference in creatinine levels between patients with or without intact/fragmented Ig (p=0.673). Analysis of overall survival in patients stratified at presentation according to uIFE results, namely the presence of intact/fragmented Ig, abnormal serum free light chain ratio-, and negative uIFE results revealed significantly shorter overall survival for the intact/fragmented Ig group (median OS: 34.5 vs. 66.0, vs. 80.6 months, respectively, p< 0.048) (figure 1). Discussion: Our findings confirm the superiority of the serum free light chain assay for detection of monoclonal free light chains as compared to urine immunofixation. However, the serum free light chain assay is inadequate for detection of intact/fragmented Ig in urine. The most important finding presented here is the observation that intact and/or fragment immunoglobulin is present in a substantial number of patients with MM. This phenomenon is mainly restricted to IgG isotypes. There are two possible explanations for these findings: first, the presence of glomerular injury, but this phenomenon (increased albumin leakage) was only seen in two patients and hence is unlikely to account for this observation. The second explanation relies upon disruption of the FcRn receptor function in immunoglobulin scavenging. This receptor will preferentially scavenge albumin in the renal setting, but dysfunction may lead to increased immunoglobulin catabolism and the presence of intact and/or fragmented Ig (Sarav, JASN, 20: 1941–1952, 2009). The results may reflect a hitherto unidentified subtle renal dysfunction. In line with this notion overall survival in our patients intact/fragmented Ig was found to be significantly shorter. Conclusion: We observed an unexpected high incidence of intact/fragmented Ig in the urine of our patients with MM. Patients with urinary excretion of intact/fragmented immunoglobulin had significantly shorter survival. These findings should be validated in further studies. Disclosures: Young: Binding Site: Employment. Harding:Binding Site: Employment.

Appraisal of IgM Kappa/IgM Lambda Variations Using HevyLite® After Rituximab As Consolidation Therapy in Patients with Waldenström's Macroglobulinemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4879-4879
Author(s):  
Jean-Richard Eveillard ◽  
Achouak Achour ◽  
Françoise N'Go Sack ◽  
Adrian Tempescul ◽  
Ronan LE Calloc'h ◽  
...  
Keyword(s):  
Partial Response ◽  
Binding Site ◽  
Light Chain ◽  
Threshold Value ◽  
Light Chains ◽  
Critical Threshold ◽  
Chop Regimen ◽  
Gamma Receptor ◽  
Significant Difference ◽  
The Impact

Abstract Abstract 4879 Background: In Waldenström's Macroglobulinemia (WM), response rates to rituximab (RTX) range from 30% to 75%. RTX is also known to generate a transient IgM flare in some patients. Our study aimed to characterize this phenomenon using the newly Hevylite® assay developed by The Binding Site that specifically quantifies IgM-kappa(K) and IgM-lambda(L). Methods: Between 2007 and 2009, 33 WM patients received a 6-course fludarabine-based regimen, 32 as first and 1 as second line. Partial response (PR), defined as a reduction ≥50% but <90% in baseline monoclonal peak (BMP) on serum protein electrophoresis (SPE), was the minimal goal. Two of the 32 patients treated at first line needed further treatment with CHOP regimen to reach the minimal PR. All received 4 consecutive weekly IV injections of 375 mg/m2 of RTX as consolidation. All were tested using the SPAplus analyzer for residual IgM-K and IgM-L at first and fourth injection. Monoclonal IgM-K or IgM-L level variations and IgM-K/IgM-L ratios were analyzed. SPE and immunofixation (IF) were performed 3 months after the fourth RTX injection to evaluate the impact of consolidation on response. Results: Of the 33 tested, at a variation threshold ≥20% relative to baseline IgM levels, 3 subgroups were defined: 15 or 45.5%, the no-flare patients, showed an increase or a decrease <20%, 11 or 33.3%, the flare patients, showed an increase ≥20% while 7 or 21.2%, the anti-flare patients, showed a decrease ≥20%. In the no-flare subgroup, there was no significant difference in monoclonal K (9) and L (6) light chains while in the 18 but 1 patients of the merged flare and anti-flare subgroups, monoclonal K light chain was overrepresented as there were 17 K vs 1 L (p<0.05). A highest initial IgM-K/L ratio (p<0.005) characterized the flare subgroup. No association was seen with demographic and hematologic characteristics and RTX pharmacokinetics. Regarding Fc gamma receptor (FcγR) IIIa-158 polymorphism, 10 patients were defined as VF and 5 as FF in the no-flare subgroup, 3 as VF, 3 as FF and 1 as VV in the anti-flare subgroup, 3 as VF and 8 as FF in the flare subgroup, with no statistically significant difference between the last 2 subgroups (p=0.22). We also analyzed the data without defining a critical threshold in monoclonal IgM level variations. Patients were, then, separated in 2 subgroups: 17 flare patients and 16 anti-flare patients. Monoclonal K light chain was overrepresented in the 2 subgroups, with 13 K vs 4 L in flare patients and 13 K vs 3 L in anti-flare patients; no statistically significant difference was found between them regarding monoclonal K and L light chains (p=0.32). No association was seen with demographic and hematologic characteristics and RTX pharmacokinetics. Regarding FcgR IIIa-158 polymorphism, there was a statistically significant difference (p=0.03) as 12 patients were of the FF subtype and 5 of the VF one in the flare subgroup while 4 were of the FF subtype, 11 of the VF one and 1 of the VV one in the anti-flare subgroup. At response evaluation 3 months after the fourth injection of RTX, all patients were screened for SPE and 22 for IF. When we compared the 17 patients of the flare subgroup to the 16 ones of the anti-flare subgroup, no patient vs 1 reached complete remission defined as disappearance of BMP on SPE and negativity of IF, 1 vs 6 advanced to very good partial response defined as a decrease ≥90% in BMP on SPE and positivity of IF and the remaining 16 vs 9 improved their partial response by a mean additional reduction of 20% (range: 0–30%) vs 18.75% (range: 0–50%) in BMP, but there was no statistically significant difference in this regard (p=0.79). Conclusions: Using Hevylite® assay, monoclonal IgM up and down variations of at least 20% following RTX occurred in up to 54.5% of our patients and this raised to 100% when no threshold value was considered. Such assay provides a reliable and highly sensitive quantitative assessment, especially in patients whose monoclonal IgM was at low concentration. Patients of the flare subgroup were predominantly of the FF subtype of FcgR IIIa-158 polymorphism while patients of the anti-flare subgroup were predominantly of VF subtype, which was statistically significant. It is tempting to hypothesize a difference in RTX mechanism of action, with ADCC predominating in the anti-flare subgroup and CDC in the flare subgroup. It would be of interest to assess such an hypothesis on a larger cohort and to analyze its impact on response profile. Disclosures: Pietrantuono: The Binding Site: Employment.

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