Presence of a membrane attack complex inhibiting protein in biological fluids

1989 ◽  
Vol 17 (4) ◽  
pp. 725-726
Author(s):  
MARIA J. WATTS ◽  
JOHN R. DANKERT ◽  
B. PAUL MORGAN
Keyword(s):  
Biological Fluids ◽  
Membrane Attack Complex ◽  
Inhibiting Protein

scholarly journals Isolation and characterization of a membrane-attack-complex-inhibiting protein present in human serum and other biological fluids

1990 ◽  
Vol 265 (2) ◽  
pp. 471-477 ◽  
Author(s):  
M J Watts ◽  
J R Dankert ◽  
B P Morgan
Keyword(s):  
Cerebrospinal Fluid ◽  
Polyclonal Antibodies ◽  
Biological Fluids ◽  
Polyacrylamide Gel Electrophoresis ◽  
Membrane Attack Complex ◽  
Erythrocyte Membranes ◽  
Dependent Manner ◽  
Isolation And Characterization ◽  
Human Erythrocyte Membranes ◽  
Inhibiting Protein

We have previously reported the isolation of a membrane-attack-complex-inhibiting protein (MIP) from human erythrocyte membranes [Watts, Patel & Morgan (1987) Complement 4, 236] and the production of polyclonal antibodies to this protein. Here we report the identification in plasma, urine, saliva and cerebrospinal fluid of a protein immunochemically identical with the membrane-derived MIP. The protein has been isolated from plasma by immunoaffinity chromatography on an anti-(erythrocyte MIP)-Sepharose column and shown by SDS/polyacrylamide-gel electrophoresis to be of similar molecular mass to the erythrocyte protein (55 kDa non-reduced and 65 kDa under reducing conditions). Monoclonal antibodies have been raised against plasma MIP and used to establish a two-site enzyme-linked immunoadsorbent assay, enabling quantification of MIP in plasma, urine and cerebrospinal fluid. Plasma MIP, though not able to incorporate spontaneously into membranes, was deposited on heterologous and homologous erythrocyte membranes during complement activation in a C8-dependent manner. Depletion of MIP from plasma resulted in enhancement of the lytic capacity of the plasma on heterologous erythrocytes.

An Unexpected Role for the Complement C5b-C9 Membrane Attack Complex (MAC) In Trafficking of Hematopoietic Stem/Progenitor Cells - a Novel Unexpected Link Between Innate Immunity and Hematopoiesis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 555-555
Author(s):  
Chihwa Kim ◽  
Wu Wan ◽  
Rui Liu ◽  
Magdalena Kucia ◽  
Janina Ratajczak ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Proteolytic Enzymes ◽  
Biological Fluids ◽  
Membrane Attack Complex ◽  
Target Cells ◽  
Complement Cascade ◽  
Hematopoietic Stem

Abstract Abstract 555 We previously reported that complement cascade (CC) is activated in bone marrow (BM) during mobilization of hematopoietic stem/progenitor cells (HSPCs; Blood 2003;101:3784; Blood 2004;103:2071; and Blood 2005;105:40) and that C5 cleavage fragments direct egress of HSPCs from BM into peripheral blood (PB) (Leukemia 2009;23:2052 and Leukemia 2010;24:976). Accordingly, C5 cleavage fragments (C5a and desArgC5a) stimulate myeloid cells in BM to secrete proteolytic enzymes and chemoattract granulocytes into peripheral blood (PB). Therefore, granulocytes form a first wave of cells that permeabilize the BM-PB endothelial barrier and prime it for subsequent egress of HSPCs. We have also observed that activation of the distal part of the complement cascade (CC), which leads to formation of C5b-C9 (also known as the membrane attack complex [MAC]), is crucial for egress/mobilization of HSPCs. It is known that proteins that form MAC can be inserted into cell membranes, resulting in cell lysis, or may remain in biological fluids as soluble MAC (sMAC) and in this “non-lytic” form may interact with target cells. We have already reported that sMAC releases bioactive lipid - sphingosine-1 phosphate (S1P) from erythrocytes, which is a major chemoattractant in mobilized peripheral blood (mPB) for HSPCs (Leukemia 2010;24:976). Since the level of sMAC increases in PB during mobilization as well as following conditioning for transplantation, we became interested in whether this protein complex affects the biology of normal HSPCs. First, we observed that, while sMAC does not affect proliferation and viability of clonogenic progenitors, it activates phosphorylation of MAPKp42/44 and AKT in both human CD34+ and murine SKL cells. Furthermore, sMAC primes and enhances chemotactic responsiveness of HSPCs to S1P and SDF-1 gradients and increases adhesiveness of these cells to BM stroma and endothelium. This effect is probably lipid raft mediated, because exposure of cells to methylo-b-cyclodextrin before chemotaxis abrogates this phenomenon. We also found that HSPCs, as well as PB mononuclear cells exposed to sMAC, secrete increased levels of PGE2 and metalloproteinases, which indicates that an increase in sMAC level in PB after conditioning for transplantation may enhance the homing properties of HSPCs. Thus, our results in toto provide novel evidence that sMAC is an underappreciated and potent regulator of HSPC trafficking and plays an important role, both direct and indirect (via released from cells S1P), in mobilization and homing of HSPCs after transplantation. In support of this notion, we found that mice displaying defects in CC activation and sMAC generation display a defect in homing of HSPCs. Thus, our data provide yet more evidence that innate immunity and the complement cascade regulate trafficking of HSPCs by (1) releasing active C3 and C5 cleavage fragments that increase the level of bioactive lipids chemoattractants in PB and BM and by (2) modulating the migratory properties of HSPCs with sMAC. We propose modulation of CC as a novel strategy for controlling both mobilization and homing of HSPCs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Isolation and characterization of the complement-inhibiting protein CD59 antigen from platelet membranes

1992 ◽  
Vol 282 (2) ◽  
pp. 409-413 ◽  
Author(s):  
B P Morgan
Keyword(s):  
Functional Characterization ◽  
Membrane Attack Complex ◽  
Cell Types ◽  
Erythrocyte Membranes ◽  
Glycosyl Phosphatidylinositol ◽  
Platelet Protein ◽  
Isolation And Characterization ◽  
Platelet Membranes ◽  
Human Erythrocyte Membranes ◽  
Inhibiting Protein

Several groups have recently described the isolation of a 20 kDa membrane-attack-complex (MAC)-inhibiting protein, termed ‘CD59 antigen’, from human erythrocyte membranes. Antibodies raised against erythrocyte CD59 antigen detect antigen on the surface of many other cell types, and in some of these cells the antigen has been shown to have a molecular mass similar to that of the erythrocyte protein and to confer resistance to lysis by the MAC. A platelet-membrane form of CD59 antigen has been described and reported to be much larger than the erythrocyte protein. Here I report the isolation of CD59 antigen from platelet membranes and its molecular and functional characterization. The platelet protein is not significantly larger than the erythrocyte form and possesses similar MAC-inhibiting activity. Platelet CD59 antigen is anchored to the membrane via a glycosyl-phosphatidylinositol link, and consequently it is suggested that deficiency of this protein might be responsible for the increased thrombotic tendency observed in paroxysmal nocturnal haemoglobinuria.

Purification of Urokinase from Complex Mixtures Using Immobilized Monoclonal Antibody Against Urokinase Light Chain

1983 ◽  
Vol 49 (01) ◽  
pp. 024-027 ◽  
Author(s):  
David Vetterlein ◽  
Gary J Calton
Keyword(s):  
Monoclonal Antibody ◽  
Light Chain ◽  
Culture Media ◽  
Biological Fluids ◽  
Single Step ◽  
High Yield ◽  
Step Method ◽  
Commercial Preparation ◽  
E Coli ◽  
Tissue Culture Media

SummaryThe preparation of a monoclonal antibody (MAB) against high molecular weight (HMW) urokinase light chain (20,000 Mr) is described. This MAB was immobilized and the resulting immunosorbent was used to isolate urokinase starting with an impure commercial preparation, fresh urine, spent tissue culture media, or E. coli broth without preliminary dialysis or concentration steps. Monospecific antibodies appear to provide a rapid single step method of purifying urokinase, in high yield, from a variety of biological fluids.

The Measurement of Heparin and Other Therapeutic Sufphated Polysaccharides in Plasma, Serum and Urine

1985 ◽  
Vol 54 (03) ◽  
pp. 630-634 ◽  
Author(s):  
J Dawes ◽  
C V Prowse ◽  
D D Pepper
Keyword(s):  
Biological Fluids ◽  
Anticoagulant Activity ◽  
Dose Range ◽  
Plasma Concentrations ◽  
Heparin Therapy ◽  
Competitive Binding Assay ◽  
First Order Kinetics ◽  
Activity Measurements ◽  
Heparin Activity ◽  
Dose Dependent

SummaryThe competitive binding assay described will specifically and accurately measure concentrations of administered heparin in biological fluids with a sensitivity of 60 ng ml-1. Neither endogenous glycosaminoglycans, nor plasma proteins such as ATIII and PF4 interfere in the assay. Semi-synthetic highly sulphated heparinoids and LMW heparin can also be measured. Using this assay heparin clearance followed simple first-order kinetics over the dose range 100-5,000 units, but the half-life was strongly dose-dependent. There was good correlation with heparin activity measurements by APTT and anti-Xa clotting assays. Plasma concentrations were measurable for at least 5 h following subcutaneous injection of 10,000 units of heparin. Excretion in the urine could be followed after all but the lowest intravenous dose. This assay, used in conjunction with measurements of heparin anticoagulant activity, will be valuable in the elucidation of mechanisms of action of heparin and the heparinoids, and in the assessment and management of problems related to heparin therapy.

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