Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal disorder characterized by hematopoietic stem cells deficient in membrane-bound glycosylphosphatidyl-(GPI)-anchored proteins due to the PIG-A mutation. The lack of certain GPI-proteins is likely responsible for the characteristic clinical features of PNH; e.g., the absence of CD59/CD55 explains intravascular hemolysis of the abnormal clone. However, no specific GPI-proteins are linked to either bone marrow failure, thrombophilia, or the inherent apoptotic resistance of PNH cells. GPI-anchored proteins have diverse functions, including maintenance of membrane and lipid raft integrity and support for transmembrane proteins. It is possible that deficiency of the GPI-anchor in the PNH clone has consequences for the expression of non-GPI-anchored proteins. For example, proteinase 3 (PR3), present in primary and secondary granules, can also be detected on the cell surface of various hematopoietic cells in its membrane-bound form (mPR3). mPR3 is linked to the expression of glycoprotein NB1 (CD177), a GPI-anchored surface receptor, whose expression parallels mPR3 levels during cell stimulation or spontaneous apoptosis. Consequently, in PNH, the absence of GPI-anchored CD177 results in a concomitant loss of mPR3 expression. We have investigated this phenomenon in greater detail in cells isolated from controls (N=20) as well as in patients with PNH (N=13). Immunoprecipitation of CD177 resulted in co-precipitation of PR3 in both cellular material (N=5) as well as in serum (N=2). Using a combination of surface and intracellular flow cytometry and Western blotting, we have demonstrated that while all granulocytes in PNH patients (N=13) and controls contain intracellular pool of PR3 (96% expression), only CD177-deficient PNH granulocytes lack mPR3 (60±15% vs. 0% in controls). As expected, the expression of mPR3 correlates with the size of PNH clone as measured by dual analysis of CD55 and CD59 in parallel with CD177. Cytokine-priming by TNF, effective in increasing mPR3 expression, failed to increase the amount of mPR3 and CD177. After isolation of detergent resistant membranes and using sucrose density-gradient centrifugation, lipid rafts from PNH patients contained expectedly lower concentrations of mPR3 than controls, but no CD177 was detected in rafts. Apart from its function in the degradation of extracellular proteins at sites of inflammation, PR3 participates in the regulation of coagulation. PR3 can down-modulate coagulation via proteolysis of the thrombin receptor, downstream of the thrombin cleavage site, thus decreasing the effects of thrombin. Unlike in granulocytes, CD177 is absent on platelets, and phopholipase C does not decrease the expression of PR3 suggesting that PR3 in platelets is not linked to a GPI-anchored chaperon. Moreover, PNH and normal platelets do not differ in the expression of mPR3. Through addition of recombinant PR3 prior to ADP- or thrombin-induced platelet activation, expression of P-selectin was greatly diminished, suggesting anticoagulant activity of PR3. Based on these results, we stipulate that decreased expression of mPR3 on PNH granulocytes and not platelets may contribute to the thrombophilic propensity seen in PNH.

Author notes

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