Oscar Berlanga

Update on collagen receptor interactions in platelets: is the two-state model still valid?

This review summarises some of the key developments that have taken place in our understanding of platelet-collagen interactions within the last 18 months. Within this time, the major activatory collagen receptor glycoprotein VI (GPVI) has been sequenced and shown to reconstitute collagen responses in a megakaryocytic cell line. It is a member of the Ig superfamily of proteins, with two extracellular Ig domains, and is constitutively associated with the Fc receptor gamma-chain (FcR gamma-chain). GPVI signals through a pathway that shares many features with those of immune receptors, with critical roles for Syk and the adapters LAT and SLP-76 in the activation of PLCgamma2. Significant developments have also taken place in regard to the role of the major adhesion receptor for collagen, the integrin alpha2beta1 (also known as GPIa-IIa). An alpha2beta1-selective collagen-based peptide has been developed and co-crystallised with the I-domain of the alpha2 subunit. Polymorphisms in alpha2 have been shown to cause wide variation in expression of alpha2beta1, with the alpha2 allele T807/A873 leading to a high level of the integrin and increased risk of stroke in young people. Activation of platelets by a wide range of agonists has been shown to increase the affinity of alpha2beta1 to intermediate or high affinity states. This has important implications for the two-site, two-state model of collagen-platelet interactions. A new model is proposed in which collagen binds initially to either alpha2beta1 or GPVI, leading to subsequent binding to the other receptor and conversion of the integrin to a high affinity state. In this model, both receptors generate intracellular signals which support platelet activation.This review summarises some of the key developments that have taken place in our understanding of platelet-collagen interactions within the last 18 months. Within this time, the major activatory collagen receptor glycoprotein VI (GPVI) has been sequenced and shown to reconstitute collagen responses in a megakaryocytic cell line. It is a member of the Ig superfamily of proteins, with two extracellular Ig domains, and is constitutively associated with the Fc receptor n -chain (FcR n -chain). GPVI signals through a pathway that shares many features with those of immune receptors, with critical roles for Syk and the adapters LAT and SLP-76 in the activation of PLC n 2. Significant developments have also taken place in regard to the role of the major adhesion receptor for collagen, the integrin f 2 g 1 (also known as GPIa-IIa). An f 2 g 1-selective collagen-based peptide has been developed and co-crystalisalised with the I-domain of the f 2 subunit. Polymorphisms in f 2 have been shown to cause wide variation in expression of f 2 g 1, with the f 2 allele T 807 /A 873 leading to a high level of the integrin and increased risk of stroke in young people. Activation of platelets by a wide range of agonists has been shown to increase the affinity of f 2 g 1 to intermediate or high affinity states. This has important implications for the two-site, two-state model of collagen-platelet interactions. A new model is proposed in which collagen binds initially to either f 2 g 1 or GPVI, leading to subsequent binding to the other receptor and conversion of the integrin to a high affinity state. In this model, both receptors generate intracellular signals which support platelet activation.

The use of snake venom toxins as tools to study platelet receptors for collagen and von Willebrand factor.

A large proportion of the biologically active proteins and peptides present within snake venoms interact with components of the haemostatic system to promote or inhibit the normal sequence of events that lead to clot formation. The venom proteins achieve their effects through interaction with various components of the coagulation cascade, endothelial matrix and platelets. Within the latter group, a number of venom proteins target the interaction of platelets with the major adhesive proteins, von Willebrand factor and collagen. The venom proteins bind either the adhesive protein itself or their receptors on the platelet surface, notably GP-Ib-IX-V and GPVI. This review discusses the substantial contribution that venom proteins have made to our understanding of the role of these two adhesive proteins and their receptors (excluding GPIIb-IIIa) in platelet regulation.

Suppression of the noninvolved pair of the myeloma isotype correlates with poor survival in newly diagnosed and relapsed/refractory patients with myeloma.

Heavy light chain (HLC) assays allow precise measurement of the monoclonal and of the noninvolved polyclonal immunoglobulins of the same isotype as the M‐protein (e.g., monoclonal IgAκ and polyclonal IgAλ in case of an IgAκ myeloma), which was not possible before. The noninvolved polyclonal immunoglobulin is termed ‘HLC‐matched pair’. We investigated the impact of the suppression of the HLC‐matched pair on outcome in 203 patients with multiple myeloma, a phenomenon that likely reflects the hosts attempt to control the myeloma clone. Severe (>50%) HLC‐matched pair suppression was identified in 54.5% of the 156 newly diagnosed patients and was associated with significantly shorter survival (45.4 vs. 71.9 months, P = 0.019). This correlation was statistically significant in IgG patients (46.4 vs. 105.1 months, P = 0.017), but not in patients with IgA myelomas (32.9 vs. 54.1 months, P = 0.498). At best response, HLC‐matched pair suppression improved only in patients with ≥VGPR, indicating partial or complete humoral immune reconstitution during remission in those with excellent response. Severe HLC‐matched pair suppression retained its prognostic impact also during follow‐up after first response. In the 47 pretreated patients with relapsed/refractory disease, a similar correlation between severe HLC suppression and survival was noted (22.8 vs. not reached, P = 0.028). Suppression of the polyclonal immunoglobulins of the other isotypes than the myeloma protein correlated neither with HLC‐matched pair suppression, nor with outcome. Multivariate analysis identified severe HLC‐matched pair suppression as independent risk factor for shorter survival, highlighting the impact of isotype specific immune dysregulation on outcome in multiple myeloma. Am. J. Hematol. 91:295–301, 2016.

Suppression of the non‐involved pair of the myeloma isotype (HLC‐matched pair) correlates with poor survival in newly diagnosed and relapsed/refractory patients with myeloma

Heavy light chain (HLC) assays allow precise measurement of the monoclonal and of the noninvolved polyclonal immunoglobulins of the same isotype as the M‐protein (e.g., monoclonal IgAκ and polyclonal IgAλ in case of an IgAκ myeloma), which was not possible before. The noninvolved polyclonal immunoglobulin is termed ‘HLC‐matched pair’. We investigated the impact of the suppression of the HLC‐matched pair on outcome in 203 patients with multiple myeloma, a phenomenon that likely reflects the hosts attempt to control the myeloma clone. Severe (>50%) HLC‐matched pair suppression was identified in 54.5% of the 156 newly diagnosed patients and was associated with significantly shorter survival (45.4 vs. 71.9 months, P = 0.019). This correlation was statistically significant in IgG patients (46.4 vs. 105.1 months, P = 0.017), but not in patients with IgA myelomas (32.9 vs. 54.1 months, P = 0.498). At best response, HLC‐matched pair suppression improved only in patients with ≥VGPR, indicating partial or complete humoral immune reconstitution during remission in those with excellent response. Severe HLC‐matched pair suppression retained its prognostic impact also during follow‐up after first response. In the 47 pretreated patients with relapsed/refractory disease, a similar correlation between severe HLC suppression and survival was noted (22.8 vs. not reached, P = 0.028). Suppression of the polyclonal immunoglobulins of the other isotypes than the myeloma protein correlated neither with HLC‐matched pair suppression, nor with outcome. Multivariate analysis identified severe HLC‐matched pair suppression as independent risk factor for shorter survival, highlighting the impact of isotype specific immune dysregulation on outcome in multiple myeloma. Am. J. Hematol. 91:295–301, 2016.

Quantification of β-region IgA monoclonal proteins - should we include immunochemical Hevylite® measurements? Point.

Abstract Accurate measurement of IgA monoclonal proteins presents a significant challenge to laboratory staff. IgA heavy/light chain (Hevylite, HLC) analysis is an alternative methodology for monoclonal protein assessment, giving an independent measure of IgAκ and IgAλ concentrations. Clonality is assessed by calculating the ratio of involved immunoglobulin to background uninvolved immunoglobulin concentrations (e.g. IgAκ/IgAλ in an IgAκ patient). Here we discuss the challenges faced by the laboratory in IgA monoclonal protein assessment, and compare the performance of Hevylite assays with electrophoresis and total IgA results. We present data which validates the use of Hevylite for response assessment: in most cases, Hevylite provides comparable response assignment to that provided by serum protein electrophoresis (SPE) and total IgA; in other cases Hevylite provides additional information, such as detection of residual disease or relapse.

Comment on “Clinical Comparisons of Two Free Light Chain Assays to Immunofixation Electrophoresis for Detecting Monoclonal Gammopathy”

The recent publication by Kim et al. [1] compares the performance of two serum free light chain (sFLC) assays, the polyclonal antibody based Freelite and the monoclonal antibody based N-Latex-FLC. We welcome the opportunity to comment on the design of the study and interpretation of results. Serum and urine electrophoresis can be used to identify monoclonal gammopathy (MG) patients with gross intact monoclonal immunoglobulin or free light chain production. However, electrophoresis assays are insensitive for the detection of patients with AL amyloidosis and nonsecretory multiple myeloma (NSMM). The introduction of the Freelite assay in 2001 [2] improved the sensitivity for detection of patients with monoclonal free light chain production. This improved sensitivity has resulted in the inclusion of Freelite in international guidelines [3–5] for screening, diagnosis, and monitoring of monoclonal gammopathies. Recently, assays utilising monoclonal antibodies for the measurement of serum free light chains have become available. The assays are calibrated to Freelite, but so far there is a paucity of data comparing the clinical performance of the assays. Kim et al. analysed samples from 63 MG (n = 100 samples) and 57 non-MG (n = 57 samples) patients. Both kappa and lambda N-Latex-FLC are calibrated to the Freelite assays [6] and therefore it is not surprising that there is concordance with results in a normal population. However, we believe there are too few clinical samples from patients with light chain multiple myeloma (LCMM) (10/63) and AL amyloidosis (2/63) for Kim et al. to make a reliable clinical comparison. There were 17 discordant results in this study (13 MG and 4 non-MG patients). It would have been informative if the authors had presented the performance of the assays in the different groups of monoclonal gammopathy patients, particularly in those with LCMM and AL amyloidosis. Specifically in LCMM populations previous studies with the monoclonal antibody based N-Latex-FLC test have failed to identify all patients [6–8]. By contrast in sixteen independent studies, including samples from 682 LCMM patients, an abnormal κ/λ sFLC ratio using the Freelite assay identified 100% samples (Table 1) [9–25]. One study [26] reported a LCMM patient missed by Freelite; however the sample was correctly identified when reanalysed using the same batch of reagent, indicating a previous analytical error (personal communication). To truly understand the concordance between the assays larger studies are required in clinically relevant populations including patients with AL amyloidosis, LCMM, and NSMM. In addition, there has only been a single study comparing the assays in patient with acute kidney injury [27]. Table 1 Publications reporting detection of LCMM patients with Freelite. 4/57 non-MG patients had an abnormal κ/λ sFLC ratio using the Freelite assay but had normal ratios using the N-Latex-FLC assay. These patients had disorders (chronic kidney disease, chronic obstructive pulmonary disease, iron deficiency anaemia, and systemic lupus erythematosus) that have previously been reported to cause a perturbation of the κ/λ sFLC ratio due to poor renal function, inflammation, or immune stimulation [28–30]. In patients with renal impairment FLC removal becomes increasingly dependent on the reticuloendothelial system. Unlike renal clearance reticuloendothelial clearance is not influenced by size of the light chains [31]; therefore the production rate of kappa FLC (approximately 2x that of lambda) exerts an influence on the κ/λ FLC ratio. Whilst there have previously been reports highlighting the difference in the performance of the N-Latex-FLC assay in patients with impaired renal function, there has been no physiological explanation for this performance [32]. The quantitative assessment of free light chains by Freelite is an important laboratory test. An abnormal ratio can be used as part of an algorithm to risk stratifying monoclonal gammopathy of undetermined significance patients. Furthermore, a ratio of >100 with a monoclonal free light chain concentration >100 mg/L was recently included in the diagnostic algorithm for patients with multiple myeloma, and an abnormal ratio is useful in understanding the depth of response in patients during the course of their disease [33–35]. Given the reliance upon the numerical values we believe there is a strong requirement for better quantitative concordance between the assays, and clearly the role of Freelite in diagnosis, stratification, and response cannot be transferred to the N-Latex-FLC assay. In summary, sample selection in this study limits interpretation but supports published data showing that differences exist between the polyclonal and monoclonal FLC assays.