Evaluation of serum and urine biomarkers for severe COVID-19.

Introduction: The new coronavirus disease, COVID-19, poses complex challenges exacerbated by several factors, with respiratory tissue lesions being notably significant among them. Consequently, there is a pressing need to identify informative biological markers that can indicate the severity of the disease. Several studies have highlighted the involvement of proteins such as APOA1, XPNPEP2, ORP150, CUBN, HCII, and CREB3L3 in these respiratory tissue lesions. However, there is a lack of information regarding antibodies to these proteins in the human body, which could potentially serve as valuable diagnostic markers for COVID-19. Simultaneously, it is relevant to select biological fluids that can be obtained without invasive procedures. Urine is one such fluid, but its effect on clinical laboratory analysis is not yet fully understood due to lack of study on its composition. Methods: Methods used in this study are as follows: total serum protein analysis; ELISA on moderate and severe COVID-19 patients' serum and urine; bioinformatic methods: ROC analysis, PCA, SVM. Results and discussion: The levels of antiAPOA1, antiXPNPEP2, antiORP150, antiCUBN, antiHCII, and antiCREB3L3 exhibit gradual fluctuations ranging from moderate to severe in both the serum and urine of COVID-19 patients. However, the diagnostic value of individual anti-protein antibodies is low, in both blood serum and urine. On the contrary, joint detection of these antibodies in patients' serum significantly increases the diagnostic value as demonstrated by the results of principal component analysis (PCA) and support vector machine (SVM). The non-linear regression model achieved an accuracy of 0.833. Furthermore, PCA aided in identifying serum protein markers that have the greatest impact on patient group discrimination. The study revealed that serum serves as a superior analyte for describing protein quantification due to its consistent composition and lack of organic salts and drug residues, which can otherwise affect protein stability.

Mannose 2, 3, 4, 5, 6-O-pentasulfate (MPS): a partial activator of human heparin cofactor II with anticoagulation potential.

Thromboembolic diseases are a major cause of mortality in human and the currently available anticoagulants are associated with various drawbacks, therefore the search for anticoagulants that have better safety profile is highly desirable. Compounds that are part of the dietary routine can be modified to possibly increase their anticoagulant potential. We show mannose 2,3,4,5,6-O-pentasulfate (MPS) as a synthetically modified form of mannose that has appreciable anticoagulation properties. An in silico study identified that mannose in sulfated form can bind effectively to the heparin-binding site of antithrombin (ATIII) and heparin cofactor II (HCII). Mannose was sulfated using a simple sulfation strategy-involving triethylamine-sulfur trioxide adduct. HCII and ATIII were purified from human plasma and the binding analysis using fluorometer and isothermal calorimetry showed that MPS binds at a unique site. A thrombin inhibition analysis using the chromogenic substrate showed that MPS partially enhances the activity of HCII. Further an assessment of in vitro blood coagulation assays using human plasma showed that the activated partial thromboplastin time (APTT) and prothrombin time (PT) were prolonged in the presence of MPS. A molecular dynamics simulation analysis of the HCII-MPS complex showed fluctuations in a N-terminal loop and the cofactor binding site of HCII. The results indicate that MPS is a promising lead due to its effect on the in vitro coagulation rate.Communicated by Ramaswamy H. Sarma.

Plasma Heparin Cofactor II Activity Is Inversely Associated with Hepatic Fibrosis of Non-Alcoholic Fatty Liver Disease in Patients with Type 2 Diabetes Mellitus.

AIMS: Thrombin exerts various pathophysiological functions by activating protease-activated receptors (PARs), and thrombin-induced activation of PARs promotes the development of non-alcoholic fatty liver disease (NAFLD). Since heparin cofactor II (HCII) specifically inactivates thrombin action, we hypothesized that plasma HCII activity correlates with the severity of NAFLD. METHODS: A cross-sectional study was conducted. Plasma HCII activity and noninvasive clinical markers of hepatic fibrosis including fibrosis-4 (FIB-4) index, NAFLD fibrosis score (NFS) and aspartate aminotransferase-to-platelet ratio index (APRI) were determined in 305 Japanese patients with type 2 diabetes mellitus (T2DM). The relationships between plasma HCII activity and the clinical markers were statistically evaluated. RESULTS: Multiple regression analysis including confounding factors showed that plasma HCII activity independently contributed to decreases in FIB-4 index (p＜0.001), NFS (p＜0.001) and APRI (p=0.004). In addition, logistic regression analysis for the prevalence of advanced hepatic fibrosis defined by the cutoff points of the clinical scores showed that plasma HCII activity was the sole and common negative factor for prevalence of advanced hepatic fibrosis (FIB-4 index: p=0.002, NFS: p=0.026 and APRI: p=0.012). CONCLUSIONS: Plasma HCII activity was inversely associated with clinical hepatic fibrosis indices including FIB-4 index, NFS and APRI and with the prevalence of advanced hepatic fibrosis in patients with T2DM. The results suggest that HCII can serve as a novel biomarker for assessment of hepatic fibrosis of NAFLD in patients with T2DM.

Plasma heparin cofactor II activity is inversely associated with albuminuria and its annual deterioration in patients with diabetes.

AIMS/INTRODUCTION: Thrombin exerts various pathophysiological functions by activating protease-activated receptors (PARs). Recent data have shown that PARs influence the development of glomerular diseases including diabetic kidney disease (DKD) by regulating inflammation. Heparin cofactor II (HCII) specifically inactivates thrombin; thus, we hypothesized that low plasma HCII activity correlates with DKD development, as represented by albuminuria. MATERIALS AND METHODS: Plasma HCII activity and spot urine biomarkers, including albumin and liver-type fatty acid-binding protein (L-FABP), were determined as the urine albumin-to-creatinine ratio (uACR) and the urine L-FABP-to-creatinine ratio (uL-FABPCR) in 310 Japanese patients with diabetes mellitus (176 males and 134 females). The relationships between plasma HCII activities and those DKD urine biomarkers were statistically evaluated. In addition, the relationship between plasma HCII activities and annual uACR changes was statistically evaluated for 201/310 patients (115 males and 86 females). RESULTS: The mean plasma HCII activity of all participants was 93.8 ± 17.7%. Multivariate-regression analysis including confounding factors showed that plasma HCII activity independently contributed to the suppression of the uACR and log-transformed uACR values (P = 0.036 and P = 0.006, respectively) but not uL-FABPCR (P = 0.541). In addition, plasma HCII activity significantly and inversely correlated with annual uACR and log-transformed uACR increments after adjusting for confounding factors (P = 0.001 and P = 0.014, respectively). CONCLUSIONS: The plasma HCII activity was inversely and specifically associated with glomerular injury in patients with diabetes. The results suggest that HCII can serve as a novel predictive factor for early-stage DKD development, as represented by albuminuria.

RNAi targeting heparin cofactor II promotes hemostasis in hemophilia A.

Hemophilia A is a hemorrhagic disease due to congenital deficiencies of coagulation factor VIII (FVIII). Studies show that hemophilia patients with anticoagulant deficiency present less severe hemorrhagic phenotypes. We aimed to find a new therapeutic option for hemophilia patients by RNA interference (RNAi) targeting heparin cofactor II (HCII), a critical anticoagulant protein inactivating the thrombin. The optimal small interfering RNA (siRNA) was conjugated to an asialoglycoprotein receptor ligand (N-acetylgalactosamine [GalNAc]-HCII), promoting targeted delivery to the liver. After administration, GalNAc-HCII demonstrated effective, dose-dependent, and persistent HCII inhibition. After 7 days, in normal mice, GalNAc-HCII reduced HCII levels to 25.04% ± 2.56%, 11.65% ± 2.41%, and 6.50% ± 1.73% with 2, 5, and 10 mg/kg GalNAc-HCII, respectively. The hemostatic ability of hemophilia mice in the GalNAc-HCII-treated group significantly improved, with low thrombus formation time in the carotid artery thrombosis models and short bleeding time in the tail-clipping assays. After repeated administration, the prolonged activated partial thromboplastin time (APTT) was reduced. A 30 mg/kg dose did not cause pathological thrombosis. Our study confirmed that GalNAc-HCII therapy is effective for treating hemophilia mice and can be considered a new option for treating hemophilia patients.

Identification and characterization of a novel isoform of heparin cofactor II in human liver.

Heparin cofactor II (HCII) is predominantly expressed in the liver and inhibits thrombin in blood plasma to influence the blood coagulation cascade. Its deficiency is associated with arterial thrombosis. Its cleavage by neutrophil elastase produces fragment that helps in neutrophil chemotaxis in the acute inflammatory response in human. In the present study, we have identified a novel alternatively spliced transcript of the HCII gene in human liver. This novel transcript includes an additional novel region in continuation with exon 3 called exon 3b. Exon 3b acts like an alternate last exon, and hence its inclusion in the transcript due to alternative splicing removes exon 4 and encodes for a different C-terminal region to give a novel protein, HCII-N. MD simulations of HCII-N and three-dimensional structure showed a unique 51 amino acid sequence at the C-terminal having unique RCL-like structure. The HCII-N protein purified from bacterial culture showed a protein migrating at lower molecular weight (MW 55 kDa) as compared to native HCII (MW 66 kDa). A fluorescence-based analysis revealed a more compact structure of HCII-N that was in a more hydrophilic environment. The HCII-N protein, however, showed no inhibitory activity against thrombin. Due to large conformational variation observed in comparison with native HCII, HCII-N may have alternate protease specificity or a non-inhibitory role. Western blot of HCII purified from large plasma volume showed the presence of a low MW 59 kDa band with no thrombin activity. This study provides the first evidence of alternatively spliced novel isoform of the HCII gene.

Recombinant dermatan sulfate is a potent activator of heparin cofactor II-dependent inhibition of thrombin.

The glycosaminoglycan dermatan sulfate (DS) is a well-known activator of heparin cofactor II-dependent inactivation of thrombin. In contrast to heparin, dermatan sulfate has never been prepared recombinantly from material of non-animal origin. Here we report on the enzymatic synthesis of structurally well-defined DS with high anticoagulant activity. Using a microbial K4 polysaccharide and the recombinant enzymes DS-epimerase 1, dermatan 4-O-sulfotransferase 1, uronyl 2-O-sulfotransferase and N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase, several new glycostructures have been prepared, such as a homogenously sulfated IdoA-GalNAc-4S polymer and its 2-O-, 6-O- and 2,6-O-sulfated derivatives. Importantly, the recombinant highly 2,4-O-sulfated DS inhibits thrombin via heparin cofactor II, approximately 20 times better than heparin, enabling manipulation of vascular and extravascular coagulation. The potential of this method can be extended to preparation of specific structures that are of importance for binding and activation of cytokines, and control of inflammation and metastasis, involving extravasation and migration.

Modulating the degree of fucosylation of fucosylated chondroitin sulfate enhances heparin cofactor II-dependent thrombin inhibition.

Fucosylated chondroitin sulfate (FCS), an unusual glycosaminoglycan with fucose side chains, is a promising anticoagulant agent. To assess the effect of its structure on anticoagulant activity, its derivatives with various degrees of fucosylation (DF), molecular weights (Mw) and sulfation patterns were prepared and characterized. Biological tests showed that their APTT (activated partial thromboplastin time) prolonging activity and intrinsic factor Xase complex (factor IXa-VIIIa-Ca2+-PL complex) inhibitory activity were both reduced in FCS derivatives with lower Mw and DF. However, FCSs with DF at least 16% resulted in greater heparin cofactor II (HCII)-dependent thrombin inhibitory activity in response to decreasing DF, and these activities did not depend on Mw (Mw > 5.2 kDa). Solution competition binding assay further suggested that modulating the DF of FCS derivatives might enhance inhibition of thrombin by activating HCII. These findings imply that FCS derivatives with suitable chain length and DF value may be novel anticoagulants by activating HCII.

Alpha-2-macroglobulin and heparin cofactor II and the vulnerability of carotid atherosclerotic plaques: An iTRAQ-based analysis.

Rupture of carotid atherosclerotic plaque may cause stroke, while few biomarker in clinic can evaluate carotid plaque vulnerability. In this study, we divided the recruited participants into no plaque, stable plaque, and vulnerable plaque group according to carotid ultrasonography, and screened the differentially expressed proteins in plasma of these participants using isobaric tags for relative and absolute quantitation labeling coupled with liquid chromatography-tandem mass spectrometry. 28 proteins were identified differentially expressed, among which alpha-2-macroglobulin (α2M) and heparin cofactor II (HCII) were found to be at hub position in the interactions of these proteins by STRING analysis and were selected for enzyme-linked immunosorbent assay measurement to assess their relevance with carotid plaques vulnerability and diagnostic efficiency. The plasma level of α2M was found positively correlated, while HCII level was negatively correlated with higher vulnerability of carotid plaques. Both proteins were efficient in differentiating stable and vulnerable carotid plaques. These findings provide potential new targets for the research of carotid plaque vulnerability. Plasma α2M and HCII may be potential biomarkers for evaluation of the vulnerability of carotid plaques if further studied.

Polyphenolic-polysaccharide conjugates of Sanguisorba officinalis L. with anticoagulant activity mediated mainly by heparin cofactor II.

A macromolecular complex has been isolated from the dried flowering parts of medicinal plant Sanguisorba officinalis L. (So) by multi-step extraction procedure, including that with extraction by organic solvents to degrease the plant material, then with hot alkali, followed by neutralization, partitioning with organic solvents and dialysis. The complex was purified by size-exclusion chromatography into five fractions labeled as So1-So5. Individual fractions differed in the chemical composition and molecular weight distribution patterns. In vitro anticoagulant activity tests showed in all fractions more or less important inhibition of plasma clots, however, So3 and So4 were the most active. The anticoagulant activity of So3 was even more significant than that of the unfractionated complex So. These S. officinalis conjugates were able to inhibit mainly the activity of thrombin when they were mediated by heparin cofactor II, but what was unexpected they were the non-direct inhibitors of factor Xa, mediated by antitrombin, where such mechanism of action is typical for a highly sulphated glycosaminoglycans.

Heparin cofactor II is degraded by heparan sulfate and dextran sulfate.

Heparan sulfate normally binds to heparin cofactor II and modulates the coagulation pathway by inhibiting thrombin. However, when human heparin cofactor II was incubated with heparan sulfate, heparin cofactor II became degraded. Other glycosaminoglycans were tested, including hyaluronic acid, chondroitin sulfates, dermatan sulfate, and heparin, but only dextran sulfate also degraded heparin cofactor II. Pretreatment of heparan sulfate with heparinase reduced its heparin cofactor II-degrading activity. Heparan sulfate and dextran sulfate diminished the thrombin inhibitory activity of heparin cofactor II. Other serpins, including antithrombin III and pigment epithelium-derived factor, were also degraded by heparan sulfate. This is the first evidence of acidic polysaccharides exhibiting protein-degrading activity without the aid of other proteins.

Genetic variants and evolutionary analyses of heparin cofactor II.

Heparin cofactor II (HCII) belongs to serpin superfamily and it acts as a thrombin inhibitor in the coagulation cascade, in a glycosaminoglycan-dependent pathway using the release of a sequestered hirudin-like N-terminal tail for interaction with thrombin. This serpin belongs to multiple member group V2 of vertebrate serpin classification. However, there is no comprehensive study illustrating the exact phylogenetic history of HCII, to date. Herein, we explored phylogenetic traits of HCII genes. Structures of HCII gene from selected ray-finned fishes and lamprey varied in exon I and II with insertions of novel introns of which one in core domain for ray-finned fishes in exon II at the position 241c. We found HCII remain nested in the largest intron of phosphatidylinositol (PI) 4-kinase (PIK4CA) gene (genetic variants of this gene cause schizophrenia) at the origin of vertebrates, dated about 500MY old. We found that sequence features such as two acidic repeats (AR1-II), GAG-binding helix-D, three serpin motifs and inhibitory reactive center loop (RCL) of HCII protein are highly conserved in 55 vertebrates analyzed. We identified 985 HCII variants by analysis of 1092 human genomes with top three variation classes belongs to SNPs (84.3%), insertion (7.1%) and deletion (5.0%). We identified 37 deleterious mutations in the human HCII protein and we have described these mutations in relation to HCII sequence-structure-function relationships. These understandings may have clinical and medical importance as well.

Structure and anticoagulant activity of fucosylated glycosaminoglycan degraded by deaminative cleavage.

Fucosylated glycosaminoglycans (FGs) are complex glycosaminoglycans that exhibit potent anticoagulant activity. To study the relationship between molecular size and biological activity, oligosaccharides with (2,5)-anhydro-D-talose units at new reducing ends were prepared by hydrazine deacetylation and nitrous acid depolymerization. The product chemical structures were analyzed by one- and two-dimensional NMR methods. Additionally, anticoagulant activities were evaluated by clotting assay and chromogenic substrate cleavage. The results demonstrated that under mild deacetylation and deaminative cleavage conditions, both products were relatively homogeneous and sulfated fucose branch types and sulfate substituents remained stable. These depolymerized FGs with different molecular sizes had potent intrinsic anticoagulant activities, which were similar to those that were obtained by free-radical depolymerization with similar molecular weights. Decreasing molecular weight may weaken activity but not significantly affect factor Xase and heparin cofactor II (HCII)-mediated thrombin inhibition.