A soluble form of the glycolipid‐anchored receptor for urokinase‐type plasminogen activator is secreted from peripheral blood leukocytes from patients with paroxysmal nocturnal hemoglobinuria

Abstract

The cellular urokinase‐type plasminogen‐activator (uPA) receptor (uPAR) is a glycolipid‐anchored membrane protein thought to be involved in pericellular proteolysis during cell migration and tumor invasion. In the present study, we have identified and characterized two soluble forms of uPAR which have retained their ligand‐binding capability. One variant was generated in vitro by treatment of intact normal cells with either a phosphatidylinositol‐specific phospholipase C (PLC) or endoproteinase Asp‐N. The other soluble uPAR variant was secreted in vivo from peripheral blood leukocytes affected by the stem‐cell disorder paroxysmal nocturnal hemoglobinuria (PNH), and was found in the plasma from these PNH patients as well as in the conditioned medium from cultured PNH leukocytes. Under normal conditions, we find no evidence for any shedding or secretion of a soluble uPA‐binding counterpart to human uPAR in plasma. Unlike normal leukocytes, the PNH‐affected cells do not express uPAR on the cell surface, although they do contain apparently normal levels of uPAR‐specific mRNA. The secreted uPAR derived from PNH cells has a mobility in SDS/PAGE that is slightly higher than that of uPAR solubilized by PtdIns‐specific PLC or detergent, but resembles that of a truncated, recombinant uPAR variant, which has its C‐terminus close to the proposed glycolipid‐attachment site, suggesting that the secreted protein has been proteolytically processed for glycolipid attachment. The presence in plasma from PNH patients of such a secreted, hydrophilic form of uPAR lends support to the hypothesis that the lesion underlying the PNH disorder resides either in glycolipid biosynthesis or in the function of an as‐yet‐unidentified trans‐amidating enzyme assumed to cleave and assemble the truncated uPAR with the preformed glycolipid moiety.

Prognostic impact of urokinase-type plasminogen activator receptor (uPAR) in cytosols and pellet extracts derived from primary breast tumours

“…The presence of uPAR in the cytosolic samples prepared with a detergent-free, neutral extraction buffer, most probably represents a water-soluble form of native uPAR which has lost its glycolipid membrane anchor. As has been suggested earlier, soluble isoforms of membrane proteins like uPAR may be generated by alternative RNA splicing or by cleavage of a region between the membrane anchor and the mature integral membrane protein by limited proteolysis (Ploug et al, 1992). Alternatively spliced mRNA variants have been identified and characterized in both human and mouse cell lines and tissues (Kristensen et al, 1991;Pyke et al, 1993).…”

“…Therefore, a more plausible explanation for its presence in cytosols may be related to the pericellular proteolytic activities mediated by uPAR during cancer invasion and metastasis. Under these conditions, uPAR is likely to be exposed to a proteolytic micro-environment in vivo where it may be solubilized by phospholipases or proteases, like plasmin, and even may enter the bloodstream, as has been implied by several authors (Ploug et al, 1992;Grøndahl-Hansen et al, 1995;Pappot et al, 1997;Stephens et al, 1997). Indeed, recent publications have shown that by measuring the soluble form of uPAR in peripheral blood important prognostic information in patients with colon (Stephens et al, 1999) or ovarian cancer (Sier et al, 1998) can be obtained.…”

Procoagulant properties of microparticles released from red blood cells in paroxysmal nocturnal haemoglobinuria

“…The most important factor related to the poor prognosis of PNH is the complication of visceral thrombosis, cerebrovascular thromboembolism and pulmonary embolism (Ziakas et al, 2008). Several factors involved in thrombogenesis in PNH have been proposed: (i) chronic haemolysis, (ii) impaired fibrinolytic system, (iii) microparticles (MPs) released from injured platelets and vascular endothelia (Ploug et al, 1992;Wiedmer et al, 1993;Ninomiya et al, 1999;Simak et al, 2004). Complement (C¢) sensitivity of PNH red blood cells (RBC) is due to a deficiency in the expression of glycosyl-phosphatidylinositol (GPI)-anchored membrane proteins with C¢-regulatory activity, CD55 and CD59, on PNH-affected RBC (Wilcox et al, 1991;Parker et al, 2005;Brodsky, 2008).…”

Identical twin marrow transplantation for venous thrombosis in paroxysmal nocturnal hemoglobinuria; long-term complete remission as assessed by flow cytometry

“…15 Another proposes the absence of the receptor of the urokinase-type plasminogen activator (u-PAR) on blood monocytes and granulocytes. 16 The lack of this receptor may lead to greater clot stability thereby promoting the vascular events described in PNH patients. Reagents are now available that enable GPI-anchored surface proteins to be detected at a high level of sensitivity for monitoring treated PNH patients 17 and may well supplant the classic Ham's test for diagnosis.…”