cC1q-R (calreticulin) and gC1q-R/p33: ubiquitously expressed multi-ligand binding cellular proteins involved in inflammation and infection

The first component of complement, C1, is a multi-molecular complex comprising of C1q and the Ca(2+)-dependent tetramer C1r(2)-C1s(2). The traditional role of C1q within the complex is that of recognition signal-a signal, which is instantly converted into a highly specific intramolecular proteolytic activation of the C1r(2)-C1s(2) tetramer thereby triggering activation of the classical pathway. Another important function of C1q is its ability to bind to a wide range of cell types resulting in the induction of cell-specific biological responses. These cells include polymorphonuclear leukocytes, monocytes, lymphocytes, dendritic cells, endothelial cells and platelets. Interaction of C1q with endothelial cells and platelets, for example, leads to cellular activation followed by release of biological mediators and/or expression of adhesion molecules, all of which contribute, directly or indirectly to the inflammatory process. These specific responses are mediated by the interaction of C1q with C1q binding proteins or receptors on the cell surface. To date, four types of putative C1q binding cell surface expressed proteins/receptors have been described. These include cC1q-R/CR, or calreticulin (CR), a 60 kDa protein, which is also known as collectin receptor; gC1q-R/p33, a 33 kDa homotrimeric protein; C1q-Rp (CD93), a 120 kDa, O-sialoglycoprotein; and CR1 (CD35), the receptor for C3b. Although the specific role of each of these molecules in a given C1q-mediated cellular response is yet to be worked out, all of them may, in one form or another, participate in the inflammatory processes associated with vascular or atherosclerotic lesions, autoimmune diseases, or infections. The main focus of our laboratory for the past 20 years has been to elucidate the structure and function of cC1q-R/CR and gC1q-R/p33, both of which have been isolated and characterized on the basis of their ability to bind C1q. The purpose of this article is therefore to provide an up to date overview of these two proteins with particular emphasis on their unique structural and functional features, their multi-faceted nature and most importantly their role in infection and inflammation.     

Sensitivity of three activated partial thromboplastin time reagents to coagulation factor deficiencies

Three activated partial thromboplastin time (APTT) reagent test systems, General Diagnostics Automated APTT, American Dade Actin FS, and Pacific Hemostasis (Thromboscreen KAPTT) reagent, containing different activators for the APTT assay, were evaluated for their precision and sensitivity to factor deficiencies in the intrinsic coagulation system. The data suggest that micronized silica and ellagic acid reagent systems were similar in sensitivity to Factor VIII, X, and XII deficiencies, whereas, the micronized kaolin reagent was significantly less sensitive to these deficiencies. Factor XI deficiency was detected equally well with the use of all three reagent systems. The ellagic acid reagent was somewhat more sensitive to Factor IX deficiency than the micronized silica reagent, and the micronized kaolin reagent was again least sensitive. Both the micronized silica and ellagic acid based reagents were insensitive to all but severe deficiencies in prekallikrein, whereas the micronized kaolin reagent was unable to detect this deficiency. All three reagents were insensitive to all but severe deficiencies in high-molecular-weight kininogen. The authors conclude that the reagent systems tested, containing micronized silica or ellagic acid as activators, are similar in sensitivity when used in a routine activated partial thromboplastin time to screen for factor deficiencies, whereas the reagent system containing micronized kaolin as an activator is less sensitive.     

DC-SIGN, C1q, and gC1qR form a trimolecular receptor complex on the surface of monocyte-derived immature dendritic cells

C1q modulates the differentiation and function of cells committed to the monocyte-derived dendritic cell (DC) lineage. Because the 2 C1q receptors found on the DC surface-gC1qR and cC1qR-lack a direct conduit into intracellular elements, we postulated that the receptors must form complexes with transmembrane partners. In the present study, we show that DC-SIGN, a C-type lectin expressed on DCs, binds directly to C1q, as assessed by ELISA, flow cytometry, and immunoprecipitation experiments. Surface plasmon resonance analysis revealed that the interaction was specific, and both intact C1q and the globular portion of C1q bound to DC-SIGN. Whereas IgG reduced this binding significantly, the Arg residues (162-163) of the C1q-A chain, which are thought to contribute to the C1q-IgG interaction, were not required for C1q binding to DC-SIGN. Binding was reduced significantly in the absence of Ca(2+) and by preincubation of DC-SIGN with mannan, suggesting that C1q binds to DC-SIGN at its principal Ca(2+)-binding pocket, which has increased affinity for mannose residues. Antigen-capture ELISA and immunofluorescence microscopy revealed that C1q and gC1qR associate with DC-SIGN on blood DC precursors and immature DCs. The results of the present study suggest that C1q/gC1qR may regulate DC differentiation and function through the DC-SIGN-mediated induction of cell-signaling pathways.     

Time-dependent association between platelet-bound fibrinogen and the Triton X-100 insoluble cytoskeleton

Previous studies indicated a correlation between the formation of EDTA-resistant (irreversible) platelet-fibrinogen interactions and platelet cytoskeleton formation. The present study explored the direct association of membrane-bound fibrinogen with the Triton X-100 (Sigma Chemical Co, St Louis, MO) insoluble cytoskeleton of aspirin-treated, gel-filtered platelets, activated but not aggregated with 20 mumol/L adenosine diphosphate (ADP) or 150 mU/mL human thrombin (THR) when bound fibrinogen had become resistant to dissociation by EDTA. Conversion of exogenous 125I-fibrinogen to fibrin was prevented by adding Gly-Pro-Arg and neutralizing THR with hirudin before initiating binding studies. After 60 minutes at 22 degrees C, the cytoskeleton of ADP-treated platelets contained 20% +/- 12% (mean +/- SD, n = 14) of membrane-bound 125I-fibrinogen, representing 10% to 50% of EDTA-resistant fibrinogen binding. The THR-activated cytoskeleton contained 45% +/- 15% of platelet bound fibrinogen, comprising 80% to 100% of EDTA-resistant fibrinogen binding. 125I-fibrinogen was not recovered with platelet cytoskeletons if binding was inhibited by the RGDS peptide, excess unlabeled fibrinogen, or disruption of the glycoprotein (GP) IIb-IIIa complex by EDTA-treatment. Both development of EDTA-resistant fibrinogen binding and fibrinogen association with the cytoskeleton were time dependent and reached maxima 45 to 60 minutes after fibrinogen binding to stimulated platelets. Although a larger cytoskeleton formed after platelet stimulation with thrombin as compared with ADP, no change in cytoskeleton composition was noted with development of EDTA-resistant fibrinogen binding. Examination of platelet cytoskeletons using monoclonal antibodies, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting showed the presence of only traces of GP IIb-IIIa in the cytoskeletons of resting platelets, with no detectable increases after platelet activation or development of EDTA-resistant fibrinogen binding. These data suggest that GP IIb-IIIa-mediated fibrinogen binding to activated platelets is accompanied by time-dependent alterations in platelet-fibrinogen interactions leading to the GP IIb-IIIa independent association between bound fibrinogen and the platelet cytoskeleton.     

Studies with a murine monoclonal antibody that abolishes ristocetin-induced binding of von Willebrand factor to platelets: additional evidence in support of GPIb as a platelet receptor for von Willebrand factor

A murine monoclonal antibody directed at or near a platelet membrane receptor for the von Willebrand factor was produced by the hybridoma technique. Purified F(ab')2 fragments and/or intact antibody completely blocked the agglutination of platelets induced by both ristocetin and bovine von Willebrand factor and the binding of von Willebrand factor antigen to platelets. The antibody also decreased platelet retention, prevented the reduction in platelet electrophoretic mobility caused by bovine von Willebrand factor, and decreased the serum prothrombin time. Radiolabeled F(ab')2 fragments bound to or approximately 2.5 X 10(4) sites on normal platelets with high affinity (KD or approximately 1.5 X 10(-8) M); there was no binding to platelets from 2 patients with the Bernard-Soulier syndrome. Immunoprecipitation and affinity chromatography studies indicated that the antibody binds to glycoprotein lb at a site contained on the externally oriented portion of the GPIb alpha chain (glycocalicin). An unidentified mol wt or approximately 20,000 molecule labeled by periodate/NaB3H4 coprecipitated and copurified with GPIb.     

Tissue factor pathway inhibitor-2 (TFPI-2) recognizes the complement and kininogen binding protein gC1qR/p33 (gC1qR): implications for vascular inflammation

Evidence is accumulating to suggest that TFPI-2 is involved in regulating pericellular proteases implicated in a variety of physiologic and pathologic processes including cancer cell invasion, vascular inflammation, and atherosclerosis. Recent immunohistochemical studies of advanced atherosclerotic lesions, demonstrated a similar tissue distribution for TFPI-2, High Molecular Weight Kininogen (HK), and gC1qR/p33 (gC1qR), a ubiquitously expressed, multicompartmental cellular protein involved in modulating complement, coagulation, and kinin cascades. Further studies to evaluate TFPI-2 interactions with gC1qR demonstrated direct interactions between gC1qR and TFPI-2 using immunoprecipitation and solid phase binding studies. Specific and saturable binding between TFPI-2 and gC1qR (estimated Kd: approximately 70 nM) was observed by ELISA and surface plasmon resonance (Biacore) binding assays. Binding was inhibited by antibodies to gC1qR, and was strongly dependent on the Kunitz-2 domain of TFPI-2, as deletion of this domain reduced gC1qR-TFPI-2 interactions by approximately 75%. Deletion of gC1qR amino acids 74-95, involved in C1q binding, had no effect on gC1qR binding to TFPI-2, although antibodies to this region and purified C1q both inhibited binding, most likely via allosteric effects. In contrast, HK did not affect TFPI-2 binding to gC1qR. Binding of TFPI-2 to gC1qR produced statistically significant but modest reductions in TFPI-2 inhibition of plasmin, but had no effect on kallikrein inhibition in fluid phase chromogenic assays. Taken together, these data suggest that gC1qR may participate in tissue remodeling and inflammation by localizing TFPI-2 to the pericellular environment to modulate local protease activity and regulate HK activation.     

The laboratory evaluation of platelet dysfunction

An algorithm for platelet function testing is presented in Fig. 2. The proposed algorithm takes into account the importance of a thorough clinical bleeding history and provides an integration of screening tests with more specific diagnostic assays. As our understanding of the biochemical and molecular aspects of platelet function becomes increasingly refined, it is becoming clear that current clinical testing strategies may not be adequate to identify all significant platelet function defects. The repertoire of distinct platelet agonists is increasing to allow a more discrete look at the function of isolated platelet membrane agonist receptors, especially those involved in platelet activation by ADP, thrombin, and collagen. In addition, FACS analysis of platelets, with emphasis on surface membrane glycoprotein expression, may offer a more specific and quantitative view of platelet membrane constituents and their function. Furthermore, screening tests evaluating platelet function under physiologic and pathologic flow conditions are becoming important adjuncts to in vitro analyses, and may accentuate platelet function abnormalities, not easily discerned by platelet aggregation studies. It remains to be seen whether such screening tests will better predict clinical bleeding or thrombotic risk.     

Ca+2 mobilization and fibrinogen binding of platelets refractory to adenosine diphosphate stimulation

The mechanism of adenosine diphosphate (ADP)-induced refractoriness was explored with iodine 125-labeled fibrinogen and the fluorescent Ca+2 indicator quin-2-tetraacetoxymethyl ester (quin-2). Gel-filtered platelets were rendered refractory by incubation (30 minutes, 22 degrees C) with either 10 mumol/L ADP alone or ADP and 125I-labeled fibrinogen. During the incubation period, platelets incubated with ADP alone showed an initial increase in quin-2 fluorescence, which gradually returned to baseline levels. Addition of 125I-fibrinogen to aliquots of the platelet suspension at various times during incubation showed that fibrinogen binding was normal after 1 minute but decreased to 50% in 30 minutes. According to Scatchard analysis, this decreased binding was attributed to decreased fibrinogen receptor availability, not decreased receptor affinity. Moreover, similar numbers of glycoprotein (GP) IIb-IIIa complexes remained available on platelets before and after incubation, as judged by the ability of a monoclonal antibody (10E5) directed against a complex specific epitope on GPIIb or IIIa to bind to control and refractory platelets. After incubation, platelets aggregated poorly in response to restimulation with ADP, although the amount of fibrinogen they bound (50% of normal) was sufficient to aggregate control platelets. Platelet restimulation with ADP was not accompanied by a rise in quin-2 fluorescence or exposure of additional fibrinogen receptors. Stimulation of platelets with thrombin, however, led to a rise in quin-2 fluorescence, exposure of additional fibrinogen receptors, and enhanced aggregation. Restimulation of platelets with epinephrine also increased fibrinogen receptor exposure and restored the ability of platelets to aggregate, but was accompanied by barely detectable changes in quin-2 fluorescence similar to those observed with epinephrine-treated control platelets. Platelets incubated for 30 minutes with ADP and 125I-fibrinogen also showed an initial rise in quin-2 fluorescence, which returned to baseline levels during incubation, but the amount of platelet-bound fibrinogen, normal at the onset, remained quantitatively unchanged. Much of this fibrinogen, however, no longer dissociated from platelets in the presence of ethylenediaminetetraacetic acid or apyrase, suggesting that a different type of platelet-fibrinogen interaction had developed. Restimulation of these platelets with ADP was not accompanied by increased fibrinogen binding or quin-2 fluorescence and failed to elicit significant platelet aggregation.(ABSTRACT TRUNCATED AT 400 WORDS)