Plasma Antithrombin Research Articles

Hemostatic markers and the sepsis-related organ failure assessment score in patients with disseminated intravascular coagulation in an intensive care unit.

We investigated the correlation between disseminated intravascular coagulation (DIC) score and hemostatic parameters and sepsis-related organ failure assessment (SOFA) score with clinical outcome of patients with DIC in an intensive care unit (ICU). The SOFA score was markedly elevated in patients with DIC relative to patients without DIC and significantly higher in non-survivors than in survivors. Abnormalities in almost all hemostatic parameters were significant in patients with DIC, but there was no significant difference in almost all hemostatic parameters between survivors and non-survivors. However, plasma antithrombin (AT) levels were significantly lower in non-survivors than in survivors. Soluble fibrin (SF) and tissue type plasminogen activator (tPA)-plasminogen activator inhibitor-I (PAI-I) complex correlated significantly with the SOFA score, whereas AT levels correlated significantly and negatively with the SOFA score. We conclude that the SOFA score is useful for predicting outcome in DIC patients in the ICU, and that hemostatic parameters, especially plasma AT levels, are also useful markers for organ failure and clinical outcome.

Activation of coagulation occurs after electrical cardioversion in patients with chronic atrial fibrillation despite optimal anticoagulation with warfarin

Background: There is evidence for the activation of the coagulation system and a hypercoagulable state following cardioversion. The aim of the study was to determine whether electrical cardioversion in patients with chronic atrial fibrillation induced a prothrombotic state despite optimal anticoagulation. We studied the effects of electrical cardioversion on plasma levels of fibrinogen, antithrombin III, protein C and D-dimers. Methods: We studied 24 patients with chronic atrial fibrillation who were on optimal anticoagulation and were referred for electrical cardioversion. Samples of venous blood were taken 2 h pre and post cardioversion and 1 month later. Results: Plasma median concentrations of fibrinogen decreased significantly from 3.8 g/l (interquartile range 3.1–4.2 g/l) before cardioversion to 3.5 g/l (interquartile range 2.9–3.9 g/l) 2 h after cardioversion levels ( P=0.004). The fibrinogen levels at 1 month post cardioversion (3.45 g/l, interquartile range 3.1–3.9 g/l) were also significantly lower than baseline ( P=0.02). Plasma median levels of antithrombin III fell from 93.5 U/dl (interquartile range 89.3–97.0 U/dl) pre cardioversion to 89.5 U/dl (interquartile range 83.0–93.0 U/dl) 2 h after cardioversion ( P=0.001) and returned to normal by 1 month (94.0 U/dl; interquartile range 89.3–98.5 U/dl; P=0.0001). There were no significant changes in plasma median D-dimer or protein C levels at any time. Conclusions: We have demonstrated a significant fall in the plasma fibrinogen and antithrombin III levels in patients with chronic atrial fibrillation early after electrical cardioversion, indicating thrombin generation. This study suggests that there are haemostatic changes of thrombogenesis induced by cardioversion despite optimal anticoagulation with warfarin.

Decreased concentration of antithrombin after preoperative therapeutic heparin does not cause heparin resistance during cardiopulmonary bypass

Objective: To determine if preoperative heparin therapy causes an increase in the incidence of intraoperative heparin resistance by reducing the concentration of antithrombin in plasma. Design: Prospective laboratory investigation of clinical samples. Setting: Public tertiary care hospital and public pathology service. Participants: Forty-six patients undergoing cardiac surgery involving cardiopulmonary bypass. Interventions: Fourteen patients received preoperative heparin therapy (POHI group) and 32 patients were controls (CONT group). Measurements and Main Results: The concentration of antithrombin, activated coagulation time (ACT), and clinical parameters were measured at intervals. More POHI patients had on-bypass heparin resistance than CONT (43% and 3%, respectively, p < 0.01). The POHI group had a lower concentration of antithrombin than the CONT group before (80.9% and 92.6%, respectively, p < 0.01) and while on cardiopulmonary bypass (51.6% and 57.5%, respectively, p = 0.04). Comparison of heparin-resistant and heparin-responsive POHI patients showed that the concentration of antithrombin did not differ before bypass (82.4% and 79.8%, respectively, p = 0.53) or during bypass (51.8% and 51.4%, respectively, p = 0.91). In fact, antithrombin concentrations were slightly higher in the heparin-resistant POHI patients (not significant). POHI patients received more heparin than CONT patients (medians 787 U/kg and 600 U/kg, respectively, p = 0.01) and were transfused with more fresh frozen plasma on bypass ( p = 0.03). Conclusions: Preoperative heparin causes an increased incidence of heparin resistance and reduced antithrombin concentrations. However, heparin resistance was not causally related to reduced antithrombin because antithrombin concentrations were not different between heparin-resistant and heparin-responsive patients in the POHI group.

Inhibition of human pancreatic proteinases by human plasma alpha2-antiplasmin and antithrombin.

Human plasma alpha1-antitrypsin inhibits human pancreatic trypsin, chymotrypsin and elastase, which are massively released into the blood stream during acute pancreatitis. To examine whether the plasma proteins of individuals with genetic deficiency of alpha1-antitrypsin are protected against the deleterious action of these enzymes by other inhibitors, we have tested their inhibition by alpha2-antiplasmin and antithrombin. We have determined the inhibition rate constants kass and calculated d(t), the in vivo inhibition time. Surprisingly, trypsin is inhibited faster by alpha2-antiplasmin [kass=2.5 x 10(6) M(-1)S(-1), d(t)=2.3 s] and antithrombin [kass=1.7 x 10(5) M(-1)s(-1), d(t)=5.8 s] than by alpha1-antitrypsin [d(t)=17 s or 116 s in alpha1-antitrypsin-sufficient or alpha1-antitrypsin-deficient individuals, respectively]. Low molecular weight heparin accelerates the inhibition of trypsin by antithrombin by a factor of 16 [d(t)=0.36 s]. Antithrombin and alpha2-antiplasmin are not physiological inhibitors of chymotrypsin and elastase. These enzymes are, however, physiologically inhibited by alpha1-antitrypsin and alpha1-antichymotrypsin even in alpha1-antitrypsin-deficient individuals. We conclude that (i) low molecular weight heparin may be helpful in the management of acute pancreatitis, and (ii) genetically determined alpha1-antitrypsin deficiency probably does not lead to a significantly increased risk of plasma protein degradation during this disease.

Practical genetics: alpha-1-antitrypsin deficiency and the serpinopathies.

Alpha-1-antitrypsin (alpha(1)-antitrypsin) is the archetypal member of the serine proteinase inhibitor or serpin superfamily. The most common severe deficiency variant is the Z allele, which results in the accumulation of mutant protein within hepatocytes. This 'protein overload' causes neonatal hepatitis, cirrhosis and hepatocellular carcinoma. The lack of circulating plasma alpha(1)-antitrypsin results in early-onset panlobular emphysema. The mechanism underlying the deficiency of Z alpha(1)-antitrypsin is due to an aberrant conformational transition within the protein and the formation of chains of polymers that tangle within the secretory pathway of hepatocytes. This mechanism also underlies the plasma deficiency of other members of the serpin superfamily to cause a class of diseases called the serpinopathies. Specifically mutant alleles of antithrombin, C1-inhibitor and alpha(1)-antichymotrypsin have been reported that favour the spontaneous formation of polymers and the retention of protein within hepatocytes. The consequent lack of plasma antithrombin, C1-inhibitor and alpha(1)-antichymotrypsin results in thrombosis, angio-oedema and emphysema, respectively. Moreover, the polymerisation of mutants of neuroserpin results in the retention of polymers within neurones to cause the inclusion body dementia, familial encephalopathy with neuroserpin inclusion bodies or FENIB. We review here the genetic and molecular basis and clinical features of alpha(1)-antitrypsin deficiency, and show how this provides a platform to understand the other serpinopathies.

Anticoagulant activity of fucoidan from brown algae Fucus evanescens of the Okhotsk Sea.

In vitro and in vivo experiments showed that anticoagulant activity of sulfated polysaccharide from Fucus evanescens (brown algae of the Okhotsk Sea) was similar to that of heparin. Anticoagulant properties of fucoidan are determined by thrombin inhibition mediated via plasma antithrombin III.

Glycosaminoglycans affect the interaction of human plasma kallikrein with plasminogen, factor XII and inhibitors.

Human plasma kallikrein, a serine proteinase, plays a key role in intrinsic blood clotting, in the kallikrein-kinin system, and in fibrinolysis. The proteolytic enzymes involved in these processes are usually controlled by specific inhibitors and may be influenced by several factors including glycosaminoglycans, as recently demonstrated by our group. The aim of the present study was to investigate the effect of glycosaminoglycans (30 to 250 micro/ml) on kallikrein activity on plasminogen and factor XII and on the inhibition of kallikrein by the plasma proteins C1-inhibitor and antithrombin. Almost all available glycosaminoglycans (heparin, heparan sulfate, bovine and tuna dermatan sulfate, chondroitin 4- and 6-sulfates) reduced (1.2 to 3.0 times) the catalytic efficiency of kallikrein (in a nanomolar range) on the hydrolysis of plasminogen (0.3 to 1.8 microM) and increased (1.9 to 7.7 times) the enzyme efficiency in factor XII (0.1 to 10 microM) activation. On the other hand, heparin, heparan sulfate, and bovine and tuna dermatan sulfate improved (1.2 to 3.4 times) kallikrein inhibition by antithrombin (1.4 microM), while chondroitin 4- and 6-sulfates reduced it (1.3 times). Heparin and heparan sulfate increased (1.4 times) the enzyme inhibition by the C1-inhibitor (150 nM).

Raloxifene decreases plasma antithrombin activity

Raloxifene decreases plasma antithrombin activity

Raloxifene decreases plasma antithrombin activity

Raloxifene decreases plasma antithrombin activity

Effects of raloxifene therapy on the anticoagulant system in postmenopausal women

Background: Raloxifene therapy is associated with a three-fold increase in the risk for venous thromboembolism; however, its effects on the hemostatic system in postmenopausal women have not been well defined. Objective: To determine the effects of raloxifene therapy on the levels of natural anticoagulant proteins in postmenopausal women. Methods: Sixteen healthy postmenopausal women were enrolled in this prospective longitudinal study. The patients were treated with raloxifene hydrochloride (60 mg/day) for a period of 6 months. Antithrombin and protein C activities and protein S antigen levels were measured in all users at baseline, and after 1, 3 and 6 months of treatment. Statistical analysis included one-way analysis of variance (ANOVA) and the Bonferroni test for multiple comparisons among the study periods. Results: Statistically significant 5.1% and 6.5% reductions of plasma antithrombin activity were observed at 3 and 6 months of therapy, respectively (p < 0.05). Compared with baseline, raloxifene did not significantly affect protein C activity or protein S level. Conclusions: The results of this prospective study show for the first time that raloxifene use is associated with a significant reduction in plasma antithrombin activity. This effect may contribute to a procoagulant state and partly explain the increased risk of venous thromboembolism in raloxifene users.

Novel approach for preparation of heparins specific to factor Xa using affinity chromatography coupled with synthetic antithrombin III-related peptides

In the blood coagulation cascade, heparin activates human plasma antithrombin III (hAT III), resulting in the inhibition of factor Xa. This polysaccharide also exhibits hemorrhagic tendency mediated by the inhibition of thrombin in heparinotherapy. Therefore, attention has focused on the development of low molecular weight heparins (LMW-heparins) that inhibit factor Xa but not thrombin. In this investigation, we examined the biochemical and physicochemical properties of hAT III-derived heparin-binding peptides (HBPs). Of all the tested HBPs, hAT III (123–139) exhibited the highest affinity with heparin and showed an inhibitory effect on the heparin-induced enhancement of hAT III activity toward factor Xa, indicating that hAT III (123–139) specifically interacts with the active region in heparin. We prepared a synthetic hAT III (123–139)-coupled affinity chromatography system, and demonstrated that this novel affinity chromatography is useful for fractionation of highly active moieties in LMW-heparins.

Exploring New Non‐Sugar Sulfated Molecules as Activators of Antithrombin.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Deletion of P1 Arginine in a Novel Antithrombin Variant (Antithrombin London) Abolishes Inhibitory Activity but Enhances Heparin Affinity and Is Associated with Early Onset Thrombosis

A novel variant of antithrombin, the major serpin inhibitor of coagulation proteases, has been identified in a patient with early onset thrombosis and abnormal plasma antithrombin activity. Sequencing of the antithrombin genes of the patient revealed that one of the two alleles was abnormal due to an in-frame deletion of the codon for the P1 arginine residue. The abnormal antithrombin was separated from the normal inhibitor by complexing the latter with thrombin followed by heparin-agarose affinity chromatography. The purified variant, antithrombin London, was completely inactive as a thrombin or factor Xa inhibitor even after heparin activation. Surprisingly, the variant bound heparin with a K(D) reflecting an approximately 10-fold greater affinity than the normal inhibitor. Stopped-flow kinetic analysis showed that this was almost entirely due to a more favorable conformational activation of the variant than the normal inhibitor, as reflected by a decreased rate constant for reversal of the activation. Consistent with its higher than normal heparin affinity, the inactive antithrombin variant was a potent competitive antagonist of the heparin-catalyzed reaction of normal antithrombin with thrombin but did not affect the uncatalyzed reaction. These results suggest that deletion of the antithrombin P1 residue partially activates the serpin by inducing strain in the reactive center loop, which destabilizes the native loop-buried state and favors the activated loop-exposed state with high heparin affinity. The unusually severe thrombosis associated with the heterozygous mutation may be explained by the ability of antithrombin London to bind endogenous heparan sulfate or heparin molecules with high affinity and to thereby block activation of the normal inhibitor.

Exploring new non-sugar sulfated molecules as activators of antithrombin

New non-sugar, small, sulfated molecules, based on our de novo rationally designed activator (−)-epicatechin sulfate (ECS), were investigated to bind and activate antithrombin, an inhibitor of plasma coagulation enzyme factor Xa. For the activators studied, the equilibrium dissociation constant ( K D) of the interaction with plasma antithrombin varies nearly 53-fold, with the highest affinity of 1.8 μM observed for morin sulfate, while the acceleration in factor Xa inhibition varies 2.6-fold. The results demonstrate that antithrombin binding and activation is a common property of these small sulfated molecules and suggests plausible directions for designing more potent activators.

Effects of raloxifene therapy on the anticoagulant system in postmenopausal women

Background: Raloxifene therapy is associated with a three-fold increase in the risk for venous thromboembolism; however, its effects on the hemostatic system in postmenopausal women have not beenwell defined.Objective: To determine the effects of raloxifene therapy on the levels of natural anticoagulant proteins in postmenopausal women.Methods: Sixteen healthy postmenopausalwomen were enrolled in this prospective longitudinal study. The patients were treated with raloxifene hydrochloride (60 mg/day) for a period of 6 months. Antithrombin and protein C activities and proteinS antigen levels were measured in all users at baseline, and after 1, 3 and 6 months of treatment. Statistical analysis included one-way analysis of variance (ANOVA) and the Bonferroni test for multiplecomparisons among the study periods.Results: Statistically significant 5.1% and 6.5% reductions of plasma antithrombin activity were observed at 3 and 6 months of therapy, respectively (p< 0.05). Compared with baseline, raloxifene did not significantly affect protein C activity or protein S level.Conclusions: The results of this prospective study show for the first timethat raloxifene use is associated with a significant reduction in plasma antithrombin activity. This effect may contribute to a procoagulant state and partly explain the increased risk of venous thromboembolismin raloxifene users.

The effects of chemotherapeutic agents on the regulation of thrombin on cell surfaces.

Thromboembolic disorders are common in cancer patients. Two major contributing factors are central venous catheters for drug delivery and the use of l-aparaginase, which decreases the plasma antithrombin level, but the causes of the hypercoagulable state in these patients are not fully understood. In this study, the T24/83 cell line was used as a model to investigate the effects of chemotherapeutic agents on cell surface thrombin regulation. Plasma thrombin generation and prothrombin consumption was increased in most of the treated cells, particularly vincristine- and adriamycin-treated cells (P < 0.05), compared with controls. However, no free thrombin generation or prothrombin consumption was observed in factor VII (FVII)-depleted plasma. No significant differences in the levels of thrombin-alpha2-macroglobulin (IIa-alpha2M) and thrombin-anti-thrombin (TAT) were observed between controls and any of the treatments, except for vincristine- and adriamycin-treated cells, which showed a significant difference in TAT production (P < 0.05). Also, there was an upregulation in tissue factor (TF) mRNA expression in etoposide-, methotrexate- and vincristine-treated monolayers compared with controls, as well as an upregulation in TF protein production in vincristine-treated cells. The data suggests that thrombin generation occurs via the extrinsic (TF-dependent) coagulation pathway on cell surfaces and that some chemotherapeutic agents are able to upregulate TF mRNA and protein expression in T24/83 cells.

Antithrombin deficiency: issues in laboratory diagnosis.

To review the current understanding of the pathophysiology of antithrombin deficiency and its role in congenital thrombophilia. Recommendations for diagnostic testing of antithrombin function and concentration, derived from the medical literature and consensus opinions of recognized experts in the field, are included. These recommendations specify whom, how, and when to test. Review of the published medical literature. A summary of the medical literature and proposed testing recommendations were prepared and presented at the College of American Pathologists Conference XXXVI: Diagnostic Issues in Thrombophilia. After discussion at the conference, consensus recommendations presented in this article were accepted after a two-thirds majority vote by the participants. Antithrombin deficiency is an infrequent genetic abnormality that may be a significant contributing cause of thrombophilia. Antithrombin deficiency also may be observed in conjunction with other genetic or acquired risk factors. Assay of antithrombin plasma levels is appropriate in the laboratory evaluation of individuals with thrombophilia, preferably using a functional, amidolytic antithrombin assay. The diagnosis of antithrombin deficiency should be established only after other acquired causes of antithrombin deficiency, such as liver disease, consumptive coagulopathy, or heparin therapy, are excluded. A low antithrombin level should be confirmed with a subsequent assay on a fresh specimen, and family studies may be helpful to establish the diagnosis. Antigenic antithrombin assays may be of benefit in subclassification of the type of antithrombin deficiency and to confirm the decreased antithrombin level in patients with type I deficiency.

Antithrombin deficiency: issues in laboratory diagnosis.

○ Objective.-To review the current understanding of the pathophysiology of antithrombin deficiency and its role in congenital thrombophilia. Recommendations for diagnostic testing of antithrombin function and concentration, derived from the medical literature and consensus opinions of recognized experts in the field, are included. These recommendations specify whom, how, and when to test. Data Sources.-Review of the published medical literature. Data Extraction and Synthesis.-A summary of the medical literature and proposed testing recommendations were prepared and presented at the College of American Pathologists Conference XXXVI: Diagnostic Issues in Thrombophilia. After discussion at the conference, consensus recommendations presented in this article were accepted after a two-thirds majority vote by the participants. Conclusions.-Antithrombin deficiency is an infrequent genetic abnormality that may be a significant contributing cause of thrombophilia. Antithrombin deficiency also may be observed in conjunction with other genetic or acquired risk factors. Assay of antithrombin plasma levels is appropriate in the laboratory evaluation of individuals with thrombophilia, preferably using a functional, amidolytic antithrombin assay. The diagnosis of antithrombin deficiency should be established only after other acquired causes of antithrombin deficiency, such as liver disease, consumptive coagulopathy, or heparin therapy, are excluded. A low antithrombin level should be confirmed with a subsequent assay on a fresh specimen, and family studies may be helpful to establish the diagnosis. Antigenic antithrombin assays may be of benefit in subclassification of the type of antithrombin deficiency and to confirm the decreased antithrombin level in patients with type I deficiency.

Observation on tissue factor pathway and some other coagulation parameters during the onset of acute cerebrocardiac thrombotic diseases

It is widely recognized that thrombosis is the major event in the evolution of acute myocardial infarction (AMI) and acute ischemic stroke (AIS). But the contribution of coagulation factors to the development of ischemic arterial diseases is still not clearly established. The goal of this study was to establish the possible relationship between coagulation factors as well as anticoagulant and the onset of AMI and AIS. The study population consisted of 69 patients with AMI and 71 with AIS as well as 50 age-matched healthy volunteers. Compared with the control group, plasma tissue factor (TF) and tissue factor pathway inhibitor (TFPI) activities and both TF and TFPI antigens were significantly higher in the AMI group; plasma TF activity and antigen in AIS group were significantly increased, but the activity and antigen of plasma TFPI were significantly decreased in the AIS group. Plasma FVII coagulation (FVII:C) activity was markedly higher in patients with AIS, but not statistically different to the control in patients with AMI. FVIII coagulation (FVIII:C) activity was remarkably higher in patients with AMI but slightly lower than the control in patients with AIS. In the AMI and AIS groups, prothrombin activity and clottable fibrinogen were significantly higher and plasma antithrombin III activity was remarkably lower than the control. The results suggested that during the onset of AMI and AIS, the initiation of TF pathway would be associated with the thrombotic events and that the blood be in hypercoagulable state. But the changes of FVII:C, TFPI and FVIII:C in AMI are different from those in AIS.

Interaction of designed sulfated flavanoids with antithrombin: lessons on the design of organic activators.

Recently, we designed (-)-epicatechin sulfate (ECS), the first small nonsaccharide molecule, as an activator of antithrombin for the accelerated inhibition of factor Xa, a key proteinase of the coagulation cascade (Gunnarsson, G. T.; Desai, U. R. J. Med. Chem. 2002, 45, 1233-1243). Although sulfated flavanoid ECS was found to bind antithrombin with an affinity ( approximately 10.7 microM) comparable to the reference trisaccharide DEF ( approximately 4.5 microM), it accelerated the inhibition of factor Xa only 10-fold as compared to the approximately 300-fold observed with DEF. To determine whether this conformational activation of the inhibitor is dependent on the structure of the organic activator and to probe the basis for the deficiency in activation, we studied the interaction of similar sulfated flavanoids with antithrombin. (+)-Catechin sulfate (CS), a chiral stereoisomer of ECS, bound plasma antithrombin with a 3-fold higher affinity (K(D) = 3.5 microM) and a 2-fold higher second-order rate constant for factor Xa inhibition (k(ACT) = 6750 M(-1) s(-1)). On the contrary, the K(D) and k(ACT) were found to be lower approximately 7.4- and approximately 2.4-fold, respectively, for its racemic counterpart, (+/-)-catechin sulfate. Dependence of the equilibrium dissociation constant on the ionic strength of the medium at pH 6.0 and 7.4 suggests that nonionic interactions contribute a major proportion ( approximately 55-73%) of the total binding energy, and only 1-2 ion pairs, in comparison to the expected approximately 4 ion pairs for the reference trisaccharide, are formed in the interaction. Competitive binding experiments indicate that activator CS does not compete with a saccharide ligand that binds antithrombin in the pentasaccharide binding site, while it competes with full-length low-affinity heparin. A molecular docking study suggests plausible binding of CS in the extended heparin binding site, which is adjacent to the binding domain for the reference trisaccharide DEF. In combination, the results demonstrate that although conformational activation of antithrombin with small sulfated flavanoids is dependent on the structure of the activator, the designed activators do not bind in the pentasaccharide binding site in antithrombin resulting in weak activation. The mechanistic investigation highlights plausible directions to take in the rational design of specific high-affinity organic antithrombin activators.