Label-free quantitative proteomics reveals fibrinopeptide B and heparin cofactor II as potential serum biomarkers in respiratory syncytial virus-infected mice treated with Qingfei oral liquid formula.

Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections. Qingfei oral liquid (QFOL), a traditional Chinese medicine, is widely used in clinical treatment for RSV-induced pneumonia. The present study was designed to reveal the potential targets and mechanism of action for QFOL by exploring its influence on the host cellular network following RSV infection. We investigated the serum proteomic changes and potential biomarkers in an RSV-infected mouse pneumonia model treated with QFOL. Eighteen BALB/c mice were randomly divided into three groups: RSV pneumonia model group (M), QFOL-treated group (Q) and the control group (C). Serum proteomes were analyzed and compared using a label-free quantitative LC-MS/MS approach. A total of 172 protein groups, 1009 proteins, and 1073 unique peptides were successfully identified. 51 differentially expressed proteins (DEPs) were identified (15 DEPs when M/C and 43 DEPs when Q/M; 7 DEPs in common). Classification and interaction network showed that these proteins participated in various biological processes including immune response, blood coagulation, complement activation, and so forth. Particularly, fibrinopeptide B (FpB) and heparin cofactor II (HCII) were evaluated as important nodes in the interaction network, which was closely involved in coagulation and inflammation. Further, the FpB level was increased in Group M but decreased in Group Q, while the HCII level exhibited the opposite trend. These findings not only indicated FpB and HCII as potential biomarkers and targets of QFOL in the treatment of RSV pneumonia, but also suggested a regulatory role of QFOL in the RSV-induced disturbance of coagulation and inflammation-coagulation interactions.

Heparin cofactor II-dependent antithrombin activity of calcium spirulan.

Calcium spirulan (Ca-SP), a novel sulfated polysaccharide isolated from the blue-green alga Spirulina platensis, enhanced the antithrombin activity of heparin cofactor II (HC II) more than 10000-fold. The apparent second-order rate constant of thrombin inhibition by HC II was calculated to be 4.2 x 10(4) M-1 min-1 in the absence of Ca-SP, and it increased in the presence of 50 micrograms/ml Ca-SP to 4.5 x 10(8) M-1 min-1. Ca-SP effectively induced the formation of a thrombin-HC II complex in plasma. In the presence of Ca-SP, both the recombinant HC II variants Lys173-->Leu and Arg 189-->His, which are defective in interactions with heparin and dermatan sulfate, respectively, inhibited thrombin in a manner similar to native rHC II. This result indicates that the binding site of HC II for Ca-SP is different from the heparin- or dermatan sulfate-binding site. When we removed the calcium from the Ca-SP, the compound did not exert any antithrombin activity. Furthermore, Na-SP, which was prepared by replacement of the calcium in Ca-SP with sodium, accelerated the antithrombin activity of HC II as Ca-SP did. We therefore suggest that the molecular conformation maintained by Ca or Na is indispensable to the antithrombin activity of Ca-SP. The HC II-dependent antithrombin activity of Ca-SP was almost totally abolished by treatment with chondroitinase AC I, heparinase or heparitinase, but not by treatment with chondroitinase ABC and chondroitinase AC II, suggesting that a heparin- or dermatan sulfate-like structure is not responsible for the activation of HC II by Ca-SP. Ca-SP is therefore thought to be a unique sulfated polysaccharide which shows a strong antithrombin effect in an exclusively HC II-dependent manner.