Human platelet-initiated formation and uptake of the C5-9 complex of human complement.

Abstract

A B S T R A C T We have studied the interaction of radiolabeled complement components with normal human platelets, platelets from a patient with paroxysmal nocturnal hemoglobinuria, and rabbit platelets in the absence of known complement activators or in the presence of coba venon factor (CVF). When unwashed platelets in platelet-rich plasma, or washed platelets suspended in serum or autologous plasma, were incubated for 30 min, C3 and terminal components (C5, C8, and C9) were found to bind to them. The terminal components were shown to be bound as the C5-9 complex, rather than as individual proteins, by eluting them from the platelet membrane and examining their behavior on ultracentrifugation. They cosedimented at a rate characteristic of the stable C5-9 complex (22S). As many as 370-1,380 C5-9 complexes/platelet were calculated to have been bound during the incubation period. The complex so formed did not differ by ultracentrifugational criteria from that binding to rabbit platelets after CVF activation of complement. Though C3 was not included in the complex, it did not appear to be bound by nonspecific absorption. It could not be removed by washing but rather was eluted by the freeze-thaw technique used to elute the C5-9 complex. Incubation of radiolabeled components in platelet-free plasma did not result in C5-9 complex formation, indicating an initiating role for platelets in this reaction. In contrast to platelets, erythrocytes incubated in analogous plasma did not induce detectable C5-9 formation. Neither EDTA, phenylmethylsulfonylfluoride, nor epsilon-amino-N-caproic acid prevented platelet-initiated formation of C5-9, suggesting that the reaction may involve mechanisms of complement activation not previously described.

Interactions of the platelets in paroxysmal nocturnal hemoglobinuria with complement. Relationship to defects in the regulation of complement and to platelet survival in vivo.

“…The data reported in this study support the observation of Zimmerman and Kolb (34) that the platelets in PNH do not take up more C3 than normal platelets when exposed to CoVFtreated serum. However, these workers reported an increased uptake ofC8, presumably in the form ofmembrane attack complex by the platelets of one PNH patient under the same conditions.…”

“…However, these workers reported an increased uptake ofC8, presumably in the form ofmembrane attack complex by the platelets of one PNH patient under the same conditions. We did not see any increase in the uptake of membrane attack complexes under our experimental conditions, which differed somewhat from those of Zimmerman and Kolb (34), particularly with regard to the presence of EDTA in the reaction mixture. In whole serum systems, the interactions ofthe platelets with activated coagulation factors, especially thrombin, as well as with complement components must be taken into consideration.…”

Neoantigen of the polymerized ninth component of complement. Characterization of a monoclonal antibody and immunohistochemical localization in renal disease.

“…Briefly, antibody-sensitized sheep erythrocytes (EA) were incubated with human serum diluted in VBS++ for 60 min at 37°C. The complement-coated EA membranes (EAC) were washed three times in BEP buffer, centrifuged at 27,000 g and 4°C for 20 min, solubilized with Triton X-100 (Sigma Chemical Co.) or Zwittergent ionic detergent [3][4][5][6][7][8][9][10][11][12] (SB12, Calbiochem-Behring, La Jolla, CA) for 30 min at room temperature. The detergent-to-protein weight ratio was 40:1.…”

“…Although the MAC has been recognized for several years on lymphocytes (10), polymorphonuclear leukocytes (11), and platelets (12), its presence in diseased tissues has been appreciated only recently. Biesecker et al (13,14) demonstrated that a polyclonal antibody to MAC fixes to immune deposits in the kidney and skin of patients with systemic lupus erythematosus (SLE).…”

Platelet Mediated Complement Activation

“…Generation of a novel thrombin dependent C5 convertase (Polly et al 1978;Polley and Nachman 1979;Zimmerman and Kolb 1976) has been described during blood coagulation, and direct activation of C5 and other complement components by thrombin has been reported (Huber-Lang 2006 and this volume).…”