suPAR MEASUREMENT, ORIGIN AND FUNCTION
suPAR Measurements
Many studies of suPAR in human are based on ELISA measurements of “bulk” suPAR (bulk-suPAR), i.e. quantifying the sum of uPAR(I-III), uPAR(II-ΠI) and uPAR(I-III)-ligand complexes.
Earlier studies only detected suP AR(I-III) and suP AR(II-III) in plasma [71] and serum [70] from healthy individuals in contrast to the recent detection of all suPAR forms (suPAR(I-III), suPAR(II-III), suPAR(I)) in normal plasma by time-resolved fluorescence immunoassay (TRFIA) [12,72,93] - a discrepancy probably attributed to better sensitivity of the newer techniques. As descript later, both suPAR(I-III), suPAR(II-III), but not suPAR(I) carries prognostic value in HIV infection. However, the combined measurement of suPAR(I- III), suPAR(II-III) carries the strongest prognostic value.
Most studies on suPAR have been carried out using in-house suPAR assay’s and suPAR values obtained from different groups are not directly comparable. In 2007, the first commercial CE/IVD approved suPAR assay (suPARnostic, ViroGates, Denmark) became available. The monoclonal-monoclonal detection assay quantifies suPAR(I-III) and suPAR(II-III) and the assay has, in contrast to previous suPAR assays, been developed on clinical value and not on best binding antibodies. This was done by developing 28 monoclonal hybridoma cell lines of anti-suPAR antibodies. These were combined into 8 ELISA’s and plasma samples from HIV infected individuals with known outcomes were analysed. While all antibody combinations gave significant prognostic value, one set of antibodies (termed VG-1 and VG-2) gave superior prognostic value and forms the basis for the suPARnostic assay. The availability of a commercial assay will allow cross-study comparison of suPAR values in different diseases.
suPAR Origin
The origin of suPAR in blood of healthy individuals is mainly unknown [94] although there are several potential sources.
Senescence of uPAR-expressing leukocytes [95], differentiation of megakaryocytes into platelets [57], hematopoietic stem cell maturation [63] or mobilization from the bone marrow [96,97], clot formation [98] and resolution [31,99], constitutive release from ECs and/or leukocytes [12,16,59,70] and leukocyte adhesion/migration [61].For long time the origin of the high circulating suPAR levels in cancer patients was unknown and only recently it was demonstrated that mice carrying transplanted human xenograft tumours have human suPAR in their blood that correlates positively with tumour volume [95]. In line with this, a recent study demonstrated that the circulating suPAR level correlates positively with the number of circulating tumour cells and the amount of uPAR in tumour cell lysates from patients with acute leukaemia [71].
The origin of the high circulating suPAR levels in patients with severe infections or inflammatory diseases is yet unknown.
suPAR Functions
Only one study has documented in vivo function of suPAR by injecting suPAR84-95 to mice where it induced leukocytosis and CD34+ hematopoietic stem/progenitor cell mobilization in magnitude similar to granulocyte-colony-stimulating factor (G-CSF) [97].
Although the function of suPAR in the blood is mainly unknown, many studies in vitro have indicated that suPAR has biological activities either by directly modulating cell adhesion and migration, by competitive inhibition of cell-bound uPAR and/or by substituting functions of cell-bound uPAR.
Modulation of Cell Adhesion and Migration
suPAR may modulate cell adhesion and migration, through binding to vitronectin and inhibition of uPAR binding, by interaction with integrins and by induction of chemotaxis. Also, interaction with cell-bound uPAR through formation of uPAR/uPAR or uPAR/suPAR dimers results in preference for lipid rafts and increased affinity for vitronectin binding [100].
suP AR(I-III) can bind to the active conformation of the β2-integrin Mac-1 (CD11b/CD18) [74] and suPAR(II-III) and suPAR(I) can bind to integrins expressed on the cell surface [86].
suPAR(I-III), suPAR(II- III) and suPAR(I) all support adhesion of integrin expressing cells [86] and suPAR(I-III) and suPAR(II-III) promote adhesion of macrophages to vitronectin and fibronectin in vitro [101]. Thus, (activated) integrins are target for direct interaction and regulation by suPAR.The chemotactic epitope of suP AR(II-III) is a strong inducer of chemotaxis in monocyte-like cells [90]. suPAR(II-III) may however rather modulate than stimulate leukocyte recruitment to sites of inflammation since suPAR(II-III) interferes with the chemokine-induced rapid integrin-dependent cell adhesion and chemotaxis [92]. Also, binding of suPAR (II-III) to the 7-transmembrane G-protein coupled receptor FPRL-1 results in desensitisation of chemokine receptors such as CCR5 [92] and thus influencing cellular trafficking.
Competitive Inhibition of Cell-Bound uPAR
In vitro, suPAR(I-III) can competitively inhibit the binding of uPA to cell-bound uPAR on haematopoietic cells [102], vascular endothelial cells [59] and various tumour cells [103,104]. By competing with cell-bound uPAR, suPAR(I-III) may function as a scavenger for uPA by inhibiting cell-associated plasminogen activation [102,104] and tumour cell proliferation and invasion in vitro [103]. In addition, tumours with overexpression of suPAR in vivo (mice carrying transplanted human xenograft breast tumours [104] or ovarian tumours [105] transfected with expression plasmids encoding suPAR) have reduced growth and colonization potential [104,105] compared to normal tumours. These findings indicate that high local levels of suPAR may restrict invasion and proliferation of tumour cells. Notably, suPAR in plasma from sepsis patients and in extra-vascular exudates contains increased uPA binding capacity [16].
Substituting Functions for Cell-Bound uPAR
suPAR(I-III) (but not suPAR(II-III)) can reconstitute integrin-associated adhesion of uPAR-deficient leukocytes [76] because suPAR(I-III) modulates integrin function similarly to cell-bound uPAR(I-III) [74].
suPAR(I-III) can also increase uPA binding in uPAR-deficient cells [59] and addition of suPAR(I-III) to tumour cells with low uPAR-expression (transfected with anti-sense uPAR mRNA) restores extracellular regulated kinase (ERK) activation which may stimulate proliferation [106]. suPAR(II-III) can induce chemotaxis in uPAR-deficient cells and it can activate the tyrosine kinase p56/p59hck in cells with normal uPAR-expression [90]. suPAR(I-III) in complex with uPA can bind to ECM-bound vitronectin and become distributed at sites distant from its production [81] and suPAR(I-III) can redistribute the presentation of uPA on the cell surface through vitronectin binding [59]. Pro-uPA-mediated fibrinolysis is enhanced by complexformation with suPAR(I-III) [31] through IgG-mediated stimulation and reduced inactivation by PAI-1 [99]. Pro-uPA-mediated fibrinolysis is not enhanced by complex-formation with suPAR(I) or suPAR(II-III) [99].Thus, suPAR may both serve as a physiological inhibitor of cell surface proteolysis by competitive inhibition of cell-bound uPAR or as an alternative non-cell-bound plasminogen activation site [68].