Heparin Cofactor II Activity Research Articles

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

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. 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. 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). 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.

1339-P: Plasma Heparin Cofactor II Activity Is Inversely Associated with the Development NAFLD in Patients with Diabetes

Aims and Introduction: The prevalence and incidence of nonalcoholic fatty liver disease (NAFLD) have been increasing worldwide. Thrombin exerts various pathophysiological functions via activation of protease-activated receptors (PARs) , and activation of PARs promotes the development of NAFLD. Since heparin cofactor II (HCII) specifically inhibits thrombin action by binding to dermatan sulfate proteoglycans in various tissue matrices, we hypothesized that plasma HCII activity is associated with the development of NAFLD. Materials and Methods: Plasma HCII activity and clinical markers for assessment of hepatic fibrosis including Fibrosis 4 (FIB-4) index, NAFLD fibrosis score (NFS) , and aspartate aminotransferase to platelet ratio index (APRI) were determined in 322 Japanese patients with diabetes mellitus (183 males and 139 females; mean age, 66.0±11.3 years) . The relationships between plasma HCII activities and the clinical markers were statistically evaluated. Results: The mean plasma HCII activity in all participants was 94.0 ± 17.7%. Multivariate regression analysis with adjustments for confounding factors including age, sex, and serum albumin showed that plasma HCII activity independently contributed to reduction of the FIB-4 index (p < 0.0001) , NFS (p < 0.0001) , and APRI (p < 0.05) . Conclusions: Plasma HCII activity was independently and inversely associated with the clinical markers of hepatic fibrosis. The results suggest that HCII can serve as a novel therapeutic target for the development of NAFLD in patients with diabetes. Disclosure T.Hara: None. I.Endo: None. M.Matsuhisa: Research Support; Nissui , Sysmex Corp., Speaker’s Bureau; Astellas Pharma Inc., Eli Lilly and Company, Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novo Nordisk, Sanofi, Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited. M.Abe: None. K.Aihara: None. H.Yamagami: None. S.Masuda: None. Y.Mitsui: None. K.Kurahashi: None. S.Yoshida: None. T.Otoda: None. T.Yuasa: None. A.Kuroda: None.

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

Aims/IntroductionThrombin 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 MethodsPlasma 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).ResultsThe 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).ConclusionsThe 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.

The Role of Heparin Cofactor Ⅱ in the Regulation of Insulin Sensitivity and Maintenance of Glucose Homeostasis in Humans and Mice.

Aim: Accelerated thrombin action is associated with insulin resistance. It is known that upon activation by binding to dermatan sulfate proteoglycans, heparin cofactor II (HCII) inactivates thrombin in tissues. Because HCII may be involved in glucose metabolism, we investigated the relationship between plasma HCII activity and insulin resistance.Methods and Results: In a clinical study, statistical analysis was performed to examine the relationships between plasma HCII activity, glycosylated hemoglobin (HbA1c), fasting plasma glucose (FPG), and homeostasis model assessment-insulin resistance (HOMA-IR) in elderly Japanese individuals with lifestyle-related diseases. Multiple regression analysis showed significant inverse relationships between plasma HCII activity and HbA1c (p = 0.014), FPG (p = 0.007), and HOMA-IR (p = 0.041) in elderly Japanese subjects. In an animal study, HCII+/+ mice and HCII+/− mice were fed with a normal diet or high-fat diet (HFD) until 25 weeks of age. HFD-fed HCII+/− mice exhibited larger adipocyte size, higher FPG level, hyperinsulinemia, compared to HFD-fed HCII+/+ mice. In addition, HFD-fed HCII+/− mice exhibited augmented expression of monocyte chemoattractant protein-1 and tumor necrosis factor, and impaired phosphorylation of the serine/threonine kinase Akt and AMP-activated protein kinase in adipose tissue compared to HFD-fed HCII+/+ mice. The expression of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase was also enhanced in the hepatic tissues of HFD-fed HCII+/− mice.Conclusions: The present studies provide evidence to support the idea that HCII plays an important role in the maintenance of glucose homeostasis by regulating insulin sensitivity in both humans and mice. Stimulators of HCII production may serve as novel therapeutic tools for the treatment of type 2 diabetes.

Abstract 18728: Plasma Heparin Cofactor II Activity is a Predictor of Diffuse Coronary Artery Narrowing in Patients with Diabetes Mellitus

Background: Heparin cofactor II (HCII) is a major physiological inhibitor of thrombin action. We previously reported high plasma HCII was protective against in-stent restenosis after coronary stenting and carotid atherosclerosis. Furthermore, we reported that HCII was a predictor of cardiovascular events in patients with coronary heart disease. Kataoka et al reported patients with DM had smaller coronary vessel and longer diseased segment than those with impaired glucose tolerance (IGT) and normal glucose tolerance. HCII and morphologic characteristics of diabetic coronary was unknown. Methods: We enrolled 20 diabetic patients examined by coronary angiography, sequentially (13 males and 7 females; age, 67.9+/-10.4yrs, HbA1c>/=7.0%). Coronary cinearteriography was analyzed by quantitative coronary angiography (QCA) with Cardiovascular Measurement System (CCIP-310/W). Segment diameter<1.5mm as diseased lesions were determined. And diseased lesion length and HCII activity were analyzed, statistically. Results: The mean plasma level of HCII activities was 101.4+/-18.6%. And the mean of diseased lesion length was 13.7+/-12.0mm. Multivariate analysis showed age, low-HDLemia, smoking habit and disease period of DM were positively correlated with ALL, and HCII activity was negatively correlated. Univariate analysis showed HCII activity was negatively correlated with ALL, and disease period of DM was positively. That is, HCII activity and disease period of DM were correlated with lesion length of coronary artery diffuse-narrowing in diabetic patients. . Conclusions: The results suggested that low level of plasma HCII activity was correlated with diffuse coronary artery narrowing in patients with diabetic mellitus.

Ascidian dermatan sulfates attenuate metastasis, inflammation and thrombosis by inhibition of P‐selectin

Cancer-associated thrombosis and enduring inflammation are strongly associated with cancer progression and metastasis. Heparin is the mostly clinically used anticoagulant/antithrombotic drug, and has recently been shown to exhibit antimetastatic and anti-inflammatory activities that are linked to inhibition of P-selectin and/or L-selectin. P-selectin-mediated platelet-tumor cell and tumor cell-endothelium interactions facilitate the initial steps of metastasis. The aim of the present study was to determine the capacity of dermatan sulfates to inhibit P-selectin and to test their potential to affect thrombosis, inflammation and metastasis in respective experimental mouse models. Two dermatan sulfates isolated from the ascidians Styela plicata and Phallusia nigra, composed of the same disaccharide core structure (IdoA2-GalNAc)(n) , but sulfated at carbon 4 or 6 of the GalNAc, respectively, have opposed heparin cofactor II (HCII) activities and are potent inhibitors of P-selectin. The ascidian dermatan sulfates effectively attenuated metastasis of both MC-38 colon carcinoma and B16-BL6 melanoma cells and the infiltration of inflammatory cells in a thioglycollate peritonitis mouse model. Moreover, both glycosaminoglycans reduced thrombus size in an FeCl(3) -induced arterial thrombosis model, irrespective of their HCII activities. The analysis of arterial thrombi demonstrated markedly reduced platelet deposition after dermatan sulfate treatment, suggesting that the glycosaminoglycan inhibited P-selectin and thereby the binding of activated platelets during thrombus formation. Collectively, these findings provide evidence that specific inhibition of P-selectin represents a potential therapeutic target in thrombosis, inflammation and metastasis, and that ascidian dermatan sulfates may serve as antiselectin agents.

Heparin Cofactor II: Discovery, Properties, and Role in Controlling Vascular Homeostasis

Heparin cofactor II (HCII) is a serine protease inhibitor (serpin) found in high concentrations in human plasma. Despite its discovery >30 years ago, its physiological function is still poorly understood. It is known to inhibit thrombin, the predominant coagulation protease, and HCII-thrombin complexes have been found in plasma, yet it is thought to contribute little to normal hemostasis. However, thrombin has several other physiological functions, and therefore many biological roles for HCII need consideration. The unique structure and mechanism of action of HCII have helped guide our understanding of HCII. In particular, HCII binds many glycosaminoglycans (GAGs) such as heparin and heparin sulfate as well as several different polyanions to enhance its inhibition of thrombin. Distinctly, HCII is able to use the GAG dermatan sulfate for accelerated thrombin inhibition. Dermatan sulfate is found in high concentrations in the walls of blood vessels as well as in placental tissue. This knowledge has led to research indicating that HCII may play a protective role in atherosclerosis and placental thrombosis. Additionally, pharmaceuticals are being developed that use the dermatan sulfate activation of HCII for anticoagulation. Although much research is still needed to fully understand HCII, this humble protein may have significant impact in our medical future. This article reviews the laboratory history, protein characteristics, structure-activity relationships, protease inhibition, physiological function, and medical relevance of HCII in hopes of regenerating interest in this sometimes forgotten serpin.

Plasma heparin cofactor II activity is inversely associated with left atrial volume and diastolic dysfunction in humans with cardiovascular risk factors

Thrombin has a crucial role in cardiac remodeling through protease-activated receptor-1 activation in cardiac fibroblasts and cardiomyocytes. As heparin cofactor II (HCII) inhibits the action of tissue thrombin in the cardiovascular system, it is possible that HCII counteracts the development of cardiac remodeling. We investigated the relationships between plasma HCII activity and surrogate markers of cardiac geometry, including left atrial volume index (LAVI), relative wall thickness (RWT) and left ventricular mass index, and deceleration time (DcT) and the ratio of peak E velocity to early diastolic mitral annulus velocity (E/e' ratio) as surrogate markers of left ventricular diastolic dysfunction measured using echocardiography in 304 Japanese elderly individuals without systolic heart failure (169 men and 135 women; mean age: 65.4 ± 11.8 years). Mean plasma HCII activity in all participants was 95.8 ± 17.0% and there was no difference between the mean plasma HCII activities in males and females. Multiple regression analysis revealed that there were significant inverse relationships between plasma HCII activity and LAVI (coefficient: -0.2302, P<0.001), between HCII activity and RWT (coefficient: -0.0007, P<0.05), between HCII activity and DcT (coefficient: -0.5189, P<0.05) and between HCII activity and E/e' ratio (coefficient: -0.0558, P<0.01). Plasma HCII activity was independently and inversely associated with the development of cardiac remodeling, including cardiac concentric change, left atrial enlargement and left ventricular diastolic dysfunction. These findings suggest that cardiac tissue thrombin inactivation by HCII is a novel therapeutic target for cardiac remodeling and atherosclerosis.

Higher specificity of the activity of low molecular weight fucoidan for thrombin-induced platelet aggregation

Algal fucoidans possess a wide variety of biological activities, including anticoagulation and antithrombosis, making them potential candidates for clinical use. We assessed the antiaggregant, anticoagulant and antithrombotic activities and the underlying mechanism of the low- and high-molecular weight fucoidans from the seaweed Laminaria japonica of Qingdao, China (F-Q and HMWF-Q). In the platelets of rats and humans, HMWF-Q demonstrated a pro-aggregation response, whereas F-Q (like the commercially purchased fucoidan (F-S) and heparin), showed an inhibitory effect on thrombin-induced aggregation with an IC 50 of 8 µg/mL, approximately five times lower than those of F-S and heparin. In the activated partial thromboplastin time test, F-Q (40 μg/mL) demonstrated less potent effect than F-S (40 μg/mL) and heparin (7 μg/mL); 162 ± 2.4 s vs. 250 ± 13.2 s and > 300 s, p < 0.01, respectively. It was also less effective than F-S on inhibiting thrombin catalyzed fibrinogen cleavage (IC 50 10 μg/mL vs. 2.8 μg/mL) in vitro and rat thrombosis in vivo at 3 mg/kg (i.v.). The inhibitory effects of F-Q and heparin on thrombin activity were strikingly enhanced by either antithrombin (AT) or heparin cofactor II (HCII). A direct interaction of F-S with thrombin, and F-Q or heparin with AT was demonstrated in both fluorescence quenching and PAGE analysis. Additionally, a pro-aggregation effect and an enhancement of thrombin activity were also observed with F-S, but not with F-Q or heparin, treatment. These results indicate that F-Q inhibits thrombin via activation of AT and HCII, whereas F-S mainly interacts directly with thrombin. Importantly, F-Q shows a higher specificity for hypoaggregation and a weaker effect for anticoagulant profiles than heparin and F-S. Therefore, F-Q could be a promising candidate for the treatment of thrombosis-related cardiovascular diseases.

Heparin Cofactor II Attenuates Vascular Remodeling in Humans and Mice

Heparin cofactor II (HCII), a serine protease inhibitor (serpin), inactivates thrombin action in the subendothelial layer of the vascular wall. Because a congenitally HCII-deficient patient has been shown to have multiple atherosclerotic lesions, it is hypothesized that HCII plays a pivotal role in the development of vascular remodeling, including atherosclerosis. To clarify this issue, 3 clinical studies concerning plasma HCII activity and atherosclerosis were carried out, and results demonstrated that a higher incidence of in-stent restenosis after percutaneous coronary intervention, maximum carotid arterial plaque thickness, and prevalence of peripheral arterial disease occurred in subjects with low plasma HCII activity. Furthermore, HCII-deficient mice were generated by a gene targeting method to determine the mechanism of the vascular protective action of HCII. Because HCII(-/-) mice were embryonically lethal, we used HCII(+/-) mice and found that they manifested augmentation of intimal hyperplasia and increased thrombosis after cuff or wire injury to the femoral arteries. HCII(+/-) mice with vascular injury showed augmentation of inflammatory cytokines and chemokines and oxidative stress. These abnormal phenotypes of vascular remodeling observed in HCII(+/-) mice were almost restored by human HCII protein supplementation. HCII protects against vascular remodeling, including atherosclerosis, in both humans and mice, and plasma HCII activity might be a predictive biomarker and novel therapeutic target for the prevention of cardiovascular diseases.

Heparin Cofactor II is an Independent Protective Factor against Peripheral Arterial Disease in Elderly Subjects with Cardiovascular Risk Factors

Heparin cofactor II (HCII) specifically inactivates thrombin action at the injured vascular wall. We have reported that HCII is a protective factor against coronary in-stent restenosis and carotid atherosclerosis; however, it is unclear whether there is any correlation between plasma HCII levels and the development of peripheral arterial disease (PAD). Plasma HCII activity and the ankle brachial pressure index (ABI) were determined in 494 elderly subjects with cardiovascular risk factors. PAD was diagnosed by ABI below 0.9, and 62 subjects were diagnosed with PAD. The relationship between factors that affect cardiovascular events and the prevalence of PAD was statistically evaluated. Mean HCII activity in PAD subjects was significantly lower than in non-PAD subjects (87.5+/-19.7% v.s. 94.6+/-17.8%, p=0.009). Multivariate logistic regression analysis showed that age (odds ratio [OR]: 1.062, p=0.0016), current smoking (OR 3.028, p=0.002) and diabetes mellitus (OR 2.656, p=0.008) were independent and progressive determinants of PAD. In contrast, HCII was an independent inhibitory factor of PAD (OR: 0.982, p=0.048). Plasma HCII activity is inversely related to the prevalence of PAD. HCII may function as the sole protective factor against PAD in elderly people with cardiovascular risk factors.

Heparin Cofactor II as a Novel Vascular Protective Factor Against Atherosclerosis

Heparin cofactor II (HCII) specifically inhibits thrombin action at the site of vascular wall injury. We encountered a congenital HCII deficiency patient with advanced multiple atherosclerotic lesions. This patient led us to conduct clinical studies to examine the role of HCII against atherosclerosis. We found that the incidence of in-stent restenosis after percutaneous coronary intervention, severity of carotid atherosclerosis and prevalence of peripheral arterial disease are inversely associated with plasma HCII activity. In order to clarify the vascular protective action of HCII, we generated HCII- deficient mice by gene targeting. In contrast to a previous study, HCII(-/-) mice were embryonically lethal. In HCII(+/-) mice, accelerated intimal hyperplasia and frequent thrombosis were observed after cuff or wire injury of femoral arteries. The number of protease-activated receptor-1 (PAR-1) -positive cells and the gene expression levels of inflammatory cytokines and chemokines were increased in the thickened vascular walls of HCII(+/-) mice. The accelerated intimal hyperplasia in HCII+/- mice with vascular injury was attenuated by human HCII administration. Furthermore, HCII deficiency exaggerated aortic plaque formation with increased oxidative stress in apolipoprotein E(-/-) mice. These results demonstrate that HCII protects against thrombin-induced vascular remodeling in both humans and mice and suggest that HCII is a predictive biomarker and therapeutic target for atherosclerosis.

Plasma heparin cofactor II activity is an independent predictor of future cardiovascular events in patients after acute myocardial infarction

This study tested the hypothesis that plasma heparin cofactor II (HCII) activity independently predicts cardiovascular events in patients after acute myocardial infarction (AMI) and attempted to elucidate the role of HCII in atherothrombosis. HCII inhibits thrombin activity by binding to dermatan sulfate and has been shown to be a novel and independent risk factor for atherosclerosis. However, there is limited data on the relation between plasma levels of HCII after AMI and future cardiovascular events. A total of 110 consecutive patients (aged 63+/-11 years) with AMI were followed up for 42+/-12 months. Plasma HCII activity was determined from blood samples collected immediately after hospitalization. The primary end point was the combined occurrence of major adverse cardiovascular events (MACE), including rehospitalization because of unstable angina, nonfatal MI, revascularization with either percutaneous coronary intervention or coronary artery bypass grafting, ischemic stroke, and cardiovascular death. All patients were divided into three groups: a high-HCII group (>122%, n=35), a normal-HCII group (>98% and <or=122%, n=41), and a low-HCII group (<or=98%, n=34). The high-HCII group had reduced MACE compared with the other groups, although the difference was not significant (P=0.150). Enhanced plasma HCII activity was, however, significantly associated with decreased MACE in the nondiabetic patients (P=0.034). In a Cox multivariate regression analysis that included all patients, plasma HCII activity was an independent predictor of future MACE (P=0.029). The results indicate a potential association between plasma HCII activity and future cardiovascular events after AMI. Moreover, HCII activity seems to play a pivotal role in atherothrombosis.

Heparin Cofactor II (HCII) Levels and the Risk of Ischemic Stroke. No Deleterious Role of HCII Deficiency but Potential Protective Role of High Plasma Level

Background: Heparin cofactor II (HCII) is a thrombin inhibitor which activity is dramatically enhanced by heparin and more specifically by dermatan sulfate, another glycosaminoglycan. Its pathophysiological role remains unknown even if cases of HCII deficiency were reported in patients with a history of venous or arterial thrombosis. Recently, it was suggested that high plasma HCII level could play a protective role against atherosclerosis in elderly individuals and protect from restenosis after femoro-popliteal or coronary artery stenting. The aim of this case-control study was to investigate the significance of plasma HCII level in the development of ischemic stroke.Methods: 81 cases (44 M and 37 F, mean age of 43 years, range: 22–63) and 80 age-and sex-matched healthy controls of the same ethnic origin. HCII activity was evaluated as antithrombin dermatan sulfate cofactor activity using a chromogenic assay. Test results were expressed in arbitrary unit per mL: 1 U/mL is the activity found in 1 mL of pooled normal human plasma.Results: Plasma HCII activity was not significantly different in cases and in controls (1.04±0.17 vs. 1.00±0.23). The same applied after stratification in males and in females. The normal range for HCII, previously defined in a large cohort of healthy individuals, was between 0.60 and 1.40 U/mL. The frequency of HCII deficiency was not significantly different in cases and in controls [n=3/81 (3.7%) vs. n=2/80, (2.5%); p=1.00] and the same applied to the frequency of HCII level above 1.40 U/mL [n=0/81 (0%) vs. n=2/80 (2.5%); p=0.25]. Interestingly, HCII activity was found above 1.30 U/mL in 7 controls and in only 1 case (p<0.05), none presenting with any of pathophysiological state usually associated with increased HCII levels such as pregnancy, oral contraceptive, inflammation, infection, or acute phase of illness.Conclusions: These results suggest that HCII deficiency is not in itself a risk factor for the development of ischemic stroke. Even if the frequency of HCII level above the normal range was not statistically different in cases and in controls, the significance of a higher proportion of controls than cases presenting with a HCII level in the high end of the normal range deserves to be further evaluated in connection with a potential protective role against ischemic stroke.

The relationship between the structure of dermatan sulfate derivatives and their antithrombotic activities

To study the relationship between the structure of dermatan sulfate (DS) derivatives and their anti-thrombotic activities, DS-derived oligosaccharides (with different structures and relative molecular weight ( M r)) were prepared, and the effects of the DS-derived oligosaccharides on the activities of heparin cofactor II (HCII), activated protein C (APC), blood platelet, and vascular endothelial cells were studied. The major disaccharides of DS and polysulfated dermatan sulfate (PSDS) were IdoA-1→3-GalNAc-4-OSO 3 and IdoA-2OSO 3-1→3-GalNAc4, 6-diOSO 3, respectively. The results showed that the consequence of the thrombotic inhibitory effects of DS and its derivatives were as follows: PSDS > low molecular weight polysulfated dermatan sulfate (LPSDS) > DS. Both DS and PSDS inhibited platelet aggregation in the concentration-dependent manner, and the IC 50 value of DS and PSDS is 12.7 ± 1.3 and 28.6 ± 0.9 mg/mL, respectively. DS oligosaccharides (DSOSs) and PSDS oligosaccharides (PSDSOSs) both significantly inhibited P-selectin expression on platelet surface ( P < 0.01), while DSOSs have no different effect compared with PSDSOSs. DSOSs and PSDSOSs significantly enhanced the activity of HCII in inhibiting thrombin in the plasma. The most active PSDSOS was PSDSOS 1 with M r of 4959, which enhanced the HCII activity by 91% ( P < 0.01). The experiments on APC activity showed that DS and its derivatives enhanced APC activity. The most active PSDSOS was PSDSOS 3 with M r of 2749, which enhanced the APC activity to 331 ± 27% ( P < 0.01). DSOSs and PSDSOSs enhanced tissue plasminogen activator (t-PA) activity and reduced the plasminogen activator inhibitor (PAI) activity from cultured human umbilical vein endothelial cells (HUVEC), resulting in the ratio of t-PA/PAI going up. PSDSOSs which have the same M r as DSOSs produced more active effects in above assays, except for platelet aggregation.

Decreased heparin cofactor II activity is associated with impaired endothelial function determined by brachial ultrasonography and predicts cardiovascular events

Heparin cofactor II (HCII) could inactivate thrombin after binding to dermatan sulfate at injured arterial walls, and has been shown to be a novel and independent antiatherosclerotic factor. However, the relation between plasma HCII activity and peripheral vascular endothelial function remains unclear. A total of 199 patients (mean age, 63+/-14 years) were enrolled and followed up for a median period of 24 months. Endothelial function was assessed using brachial ultrasonography to determine endothelium dependent flow-mediated vasodilation (FMD). Cox regression analyses were conducted for the 199 subjects, with cardiovascular events being defined as myocardial infarction (MI), percutaneous coronary intervention (PCI), coronary artery bypass grafting (CABG), ischemic stroke, and peripheral artery revascularization. A total of 31 patients (16%) had cardiovascular events. Patients with cardiovascular events had significantly lower HCII activity (112+/-34 versus 127+/-34%, p=0.027) and lower antithrombin III (ATIII) activity (82+/-12 versus 88+/-13%, p=0.014) than those without events. By multivariate analysis, age (p=0.012), hsCRP (p=0.020) and HCII activity (p=0.035) were correlated with FMD. Kaplan-Meier analysis was performed and showed plasma HCII (p=0.036) and ATIII activities (p=0.005) were predictors of cardiovascular events. By Cox regression analysis, plasma HCII activity (p=0.026) could be an independent predictor of future cardiovascular events, but not ATIII. The present study demonstrates that plasma HCII activity is positively correlated with endothelial vasodilator function. Furthermore, plasma HCII activity could be a predictor of future cardiovascular events in patients with suspected coronary artery disease, suggesting its role in atherosclerosis.

Placental dermatan sulfate: isolation, anticoagulant activity, and association with heparin cofactor II

AbstractPregnancy is associated with hemostatic challenges that may lead to thrombosis. Heparin cofactor II (HCII) is a glycosaminoglycan-dependent thrombin inhibitor present in both maternal and fetal plasma. HCII activity increases during pregnancy, and HCII levels are significantly decreased in women with severe pre-eclampsia. Dermatan sulfate (DS) specifically activates HCII and is abundant in the placenta, but the locations of DS and HCII in the placenta have not been determined. We present evidence that DS is the major anticoagulant glycosaminoglycan in the human placenta at term. DS isolated from human placenta contains disaccharides implicated in activation of HCII and has anticoagulant activity similar to that of mucosal DS. Immunohistochemical studies revealed that DS is associated with fetal blood vessels and stromal regions of placental villi but is notably absent from the syncytiotrophoblast cells in contact with the maternal circulation. HCII colocalizes with DS in the walls of fetal blood vessels and is also present in syncytiotrophoblast cells. Our data suggest that DS is in a position to activate HCII in the fetal blood vessels or in the stroma of placental villi after injury to the syncytiotrophoblast layer and thereby inhibit fibrin generation in the placenta.

Anti-thrombotic Activity of Water-soluble Calix[n]arenes

The parent p-sulfonato-calix[n]arenes and six O-monosubstituted derivatives were investigated in vitro for anticoagulant activity. Different concentrations of calixarenes were tested, showing that the compound 49-mono-(2-carboxymethoxy)-5,11,17,23,29,35,41,47-octa-sulfonato-calix[8]arene (C8SMA) has a significantly strong prolongation on the activated partial thromboplastin time (APTT) and on the thrombin time (TT) than the other calixarenes. Secondly, investigation of whether the anticoagulant behaviour was via interaction with antithrombin or Heparin Cofactor II was determined. Thrombin inhibition mediated by antithrombin (AT) and Heparin Cofactor II (HCII) activation was investigated in comparison to the biological activators, Heparin (Hep) and Dermatan sulfate (DS). The results show that the 49-mono-(2-carboxymethoxy)-5,11,17,23,29,35,41,47-octa-sulfonato-calix[8]arene (C8SMA) and 5,11,17,23,29,35-hexa-sulfonato-calix[6]arene (C6S) produce activation of HCII at 500 μM comparable to that induced by DS at 100 μM. However, activation of AT by all of the investigated calixarenes is between 10 and 50 times lower than that observed in the presence of heparin. The mechanism of the anticoagulant effect of these calixarenes is as activators of HCII and not as activators of AT.

The Heparin Binding Properties of Heparin Cofactor II Suggest an Antithrombin -Like Activation Mechanism.

Heparin cofactor II(HCII) is a serpin which specifically inhibits thrombin. Like antithrombin(AT) it requires glycosaminoglycan(GAG) binding before full activation. However unlike antithrombin no specific activators have been identified. What currently prevents selective therapeutic activation of HCII is the lack of knowledge of the determinants of GAG binding specificity. We have investigated the heparin binding properties of HCII using size fractionated heparins. The affinity of heparins for HCII, ranging in size from 8 to 26 saccharide units, were determined by following change in fluorescence of the extrinsic probe TNS. The effect of heparin concentration(from 1 to 100nM) on the rate of thrombin inhibition was determined for the same heparins under psuedo first-order conditions. Based on these studies we can conclude that binding is non specific with a minimal heparin length of 13 monosaccaride units required. The observed dependence of catalytic activity on heparin length is caused entirely by the size dependence of heparin affinity for HCII. By studying affinity at different ionic strengths it is clear that binding is critically dependent on ionic strength with a very weak non-ionic contribution to binding. Rapid binding experiments were conducted under psuedo first-order conditions in heparin using a stopped-flow fluorometer. These studies of heparin binding indicate an induced- fit mechanism which involves a conformational change in HCII. The low affinity of HCII can now be understood as a failure of interactions to reduce the rate of the reverse conformational, in a manner analogous to the interaction of AT with low affinity heparin. HCII is activated by a fully allosteric mechanism which produces a 2000 fold increase in activation on heparin binding. We conclude that the heparin binding mechanism of HCII is closely analogous to that of AT, and that the design or discovery of high affinity, HCII-specific GAGs is thus feasible.

The Heparin Binding Properties of Heparin Cofactor II Suggest an Antithrombin-like Activation Mechanism

The serpin heparin cofactor II (HCII) is a glycosaminoglycan-activated inhibitor of thrombin that circulates at a high concentration in the blood. The antithrombotic effect of heparin, however, is due primarily to the specific interaction of a fraction of heparin chains with the related serpin antithrombin (AT). What currently prevents selective therapeutic activation of HCII is the lack of knowledge of the determinants of glycosaminoglycan binding specificity. In this report we investigate the heparin binding properties of HCII and conclude that binding is nonspecific with a minimal heparin length of 13 monosaccharide units required and affinity critically dependent on ionic strength. Rapid kinetics of heparin binding indicate an induced fit mechanism that involves a conformational change in HCII. Thus, HCII binds to heparin in a manner analogous to the interaction of AT with low affinity heparin. A fully allosteric 2000-fold heparin activation of thrombin inhibition by HCII is demonstrated for heparin chains up to 26 monosaccharide units in length. We conclude that the heparin-binding mechanism of HCII is closely analogous to that of AT and that the induced fit mechanism suggests the potential design or discovery of specific HCII agonists.