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.     

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)     

Reversibility of fibrinogen fragment D1 binding to human platelets: comparison with native fibrinogen.

To gain further insight into the mechanism responsible for rendering fibrinogen bound to stimulated platelets irreversible to dissociation by EDTA or excess unlabeled fibrinogen, the present study compared the reversibility of platelet interactions with fibrinogen and its plasmic degradation product, fragment D1. Like fibrinogen binding, the binding of fragment D1 became progressively less sensitive to dissociation by EDTA, PGE1, or excess unlabeled fibrinogen. Thus in the presence of EDTA, 70 +/- 19% and 55 +/- 24% (mean +/- S.D., n = 9) of bound fragment D1 failed to dissociate from platelets 60 min after stimulation with 0.15 U/ml thrombin or the combination of 5 microM ADP and 5 microM epinephrine, respectively, compared to 75 +/- 8% and 52 +/- 17% of platelet-bound, intact fibrinogen. In contrast, platelet stimulation with chymotrypsin or Zn+2 failed to support the development of irreversible fragment D1 or fibrinogen binding. Only 8 +/- 6% and 9 +/- 3% of bound fragment D1 remained associated with chymotrypsin- or Zn+2-treated platelets, respectively, compared to 7 +/- 11% and 15 +/- 6% (mean +/- S.D., n = 3) of platelet-associated fibrinogen. These observations suggest that irreversible fragment D1 and fibrinogen binding to platelets occurs by a similar mechanism that requires neither fibrinogen alpha chain 95-97 or 572-574 RGD sequences nor multivalent ligand-receptor interactions.     

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.