To examine the hypothesis that surface Pselectin-positive (degranulated) platelets are rapidly cleared from the circulation, we developed novel methods for tracking of platelets and measurement of platelet function in vivo. Washed platelets prepared from nonhuman primates (baboons) were labeled with PKH2 (a lipophilic fluorescent dye), thrombin-activated, washed, and reinfused into the same baboons. Three-color whole blood flow cytometry was used to simultaneously (i) identify platelets with a mAb directed against glycoprotein (GP)IIb-IIIa (integrin xllb183), (ii) distinguish infused platelets by their PKH2 fluorescence, and (iii) analyze platelet function with mAbs. Two hours after infusion of autologous thrombin-activated platelets (Pselectin-positive, PKH2-labeled), 95 + 1% (mean + SEM, n = 5) of the circulating PKH2-labeled platelets had become P-selectin-negative. Compared with platelets not activated with thrombin preinfusion, the recovery of these circulating PKH2-labeled, P-selectin-negative platelets was similar 24 h after infusion and only slightly less 48 h after infusion. The loss of platelet surface P-selectin was fully accounted for by a 67.1 ± 16.7 ng/ml increase in the plasma concentration of soluble P-selectin. The circulating PKIH2-labeled, P-selectinnegative platelets were still able to function in vivo, as determined by their (i) participation in platelet aggregates emerging from a bleeding time wound, (ii) binding to Dacron in an arteriovenous shunt, (iii) binding of mAb PAC1 (directed against the fibrinogen binding site on GPIIb-IIIa), and (iv) generation of procoagulant platelet-derived microparticles. In summary, (i) circulating degranulated platelets rapidly lose surface P-selectin to the plasma pool, but continue to circulate and function; and (ii) we have developed novel three-color whole blood flow cytometric methods for tracking of platelets and measurement of platelet function in vivo.P-selectin, a member of the selectin family which includes Eand L-selectin, is a cell-adhesion molecule of activated platelets and endothelial cells (1, 2). P-selectin (also known as CD62P, GMP-140, and PADGEM protein) is a component of the a granule membrane of resting platelets that is only expressed on the platelet surface membrane during and after platelet degranulation and secretion (1,2). A soluble form of P-selectin circulates in plasma (3).Platelet surface P-selectin mediates the adherence of degranulated platelets to leukocytes in vitro (4, 5) and in vivo (6). It has therefore been postulated that surface P-selectin-positive (degranulated) platelets may be rapidly cleared from the circulation by leukocytes (2,4,5,7,8). In apparent contradiction to this postulate, other investigators have reported that degranulated platelets are no more rapidly cleared from the circulation than control platelets (9, 10). Methods have not previously been available to directly address this issue.In this study, we used novel three-color whole blood flow cytometric methods for tracking of circulating degran...

SummaryReperfusion ofischemic tissue induces an acute inflammatory response that can result in necrosis and irreversible cell injury to both local vascular endothelium and parenchyma. To examine the pathogenesis of ischemia/reperfusion injury, we have used mice deficient in complement components C3, C4, or serum immunoglobulin in a hindlimb model of ischemia. We found that mice homozygous deficient in C3 or C4 were equally protected against reperfusion injury based on a significant reduction in leakage of radiolabeled albumin out of the vasculature. This demonstrates that classical pathway complement is an important factor in the initiation ofinflammarion following reperfusion. Furthermore, mice deficient in serum immunoglobulin were equally protected and this protection could be reversed by reconstitution with serum from norreal mice. Thus, this report describes a novel mechanism for reperfusion injury that involves antibody deposition and activation of complement leading to inflammation permeability. While the pathogenesis of reperfnsion injury is not completely understood, the complement system is thought to be involved since there is clear evidence of deposition of complement components C5-9 after repeffusion of hypoxic tissue (1, 2) and injury can be inhibited in part by pretreatment with soluble complement receptor type 1 (sCR1) (3-6). Weisman et al. found that pretreatment with sCR1 substantially reduced influx of neutrophils, deposition of C5-9 and myocardial infarct size in a rat model of myocardial ischemia (3). Similar studies have used sCR1 to block or reduce inflammation after reperfusion ofischemic rat hindlimb or intestine and of mouse skeletal muscle (cremaster) (4-6). Complement receptor type 1 (CR1; CD35), which is normally expressed by human erythrocytes, B lymphocytes, granulocytes, and macrophages, binds activated C3 (C3b) and C4 (C4b) leading both to dissociation of the complement system's C3 and C5 activating enzymes and to proteolytic degradation of C3b and C4b by serum factor I. When administered in a soluble form, recombinant sCR,1 functions as a highly effective inhibitor of serum complement activation (7).To clarify the role of complement and gain insight into the pathogenesis of ischemia/reperfusion injury, we have examined novel strains of mice deficient in either complement components C3 or C4 in a mouse model ofhindlimb ischemia reperfusion. Both strains of mice were equally protected from vascular injury demonstrating that injury is mediated by the classical pathway. Moreover, mice totally deficient in antibody, i.e., recombination-activation gene 2 deficient (RAG-2-/-) (8), were equally protected and protection could be abrogated by reconstitution with fresh mouse serum. These results support the hypothesis that ischemia/reperfusion injury is initiated by natural antibody binding to hypoxic endothelium and activation of the complement system. Materials and MethodsReconstitution of Antibody-d!ficient Mice. RAG2-/-mice were reconstituted by i.v. administration of 0.5 ml of pooled ...

Intestinal ischemia-reperfusion injury is dependent on complement. This study examines the role of the alternative and classic pathways of complement and IgM in a murine model of intestinal ischemia-reperfusion. Wild-type animals, mice deficient in complement factor 4 (C4), C3, or Ig, or wild-type mice treated with soluble complement receptor 1 were subjected to 40 min of jejunal ischemia and 3 h of reperfusion. Compared with wild types, knockout and treated mice had significantly reduced intestinal injury, indicated by lowered permeability to radiolabeled albumin. When animals deficient in Ig were reconstituted with IgM, the degree of injury was restored to wild-type levels. Immunohistological staining of intestine for C3 and IgM showed colocalization in the mucosa of wild-type controls and minimal staining for both in the intestine of Ig-deficient and C4-deficient mice. We conclude that intestinal ischemia-reperfusion injury is dependent on the classic complement pathway and IgM.

Reperfusion injury of ischemic tissue represents an acute inflammatory response that can cause significant morbidity and mortality. The mechanism of injury is not fully elucidated, but recent studies indicate an important role for natural antibody and the classical pathway of complement. To test the hypothesis that injury is initiated by specific IgM, we have screened a panel of IgMproducing hybridomas prepared from peritoneal cells enriched in B-1 cells. One clone, CM22, was identified that could restore pathogenic injury in RAG-1 ؊/؊ mice in an intestinal model of ischemia͞reperfusion (I͞R). In situ activation of the classical pathway of complement was evident by deposition of IgM, complement C4, and C3 in damaged tissue after passive transfer of CM22 IgM. Sequence analysis of CM22 Ig heavy and light chains showed germ-line configurations with high homology to a V H sequence from the B-1 repertoire and a VK of a known polyreactive natural IgM. These data provide definitive evidence that I͞R injury can be initiated by clonally specific natural IgM that activates the classical pathway of complement. This finding opens an avenue for identification of I͞R-specific self-antigen(s) and early prevention of injury.