Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.5 (thrombin)
 33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

It has been suggested that protamine sulfate is a poor antidote for the bleeding side-effects of low molecular weight heparins (LMWHs) in vivo, since protamine sulfate does not completely neutralize the anti-factor Xa activity of LMWHs in vitro or ex vivo. Therefore, we performed experiments to compare directly the abilities of protamine sulfate to neutralize the anticoagulant activities of the LMWH, enoxaparine, and unfractionated heparin ex vivo, with its ability to neutralize the bleeding side-effects of both compounds in vivo. Bleeding was measured as the amount of blood lost from 5 cuts made in rabbits ears before and after treatment with enoxaparine or unfractionated heparin +/- protamine sulfate. Plasma anti-factor Xa and anti-thrombin activities ex vivo, were measured chromogenically. Doses of 400 and 1,500 anti-factor Xa U/kg of heparin and enoxaparine, respectively, were required to enhance blood loss to the same extent. Protamine sulfate completely neutralized blood loss induced by both compounds, but did not neutralize the anti-factor Xa nor antithrombin activities ex vivo. We conclude that protamine sulfate is an effective antidote for the bleeding side-effects of enoxaparine and unfractionated heparin, despite its inability to completely neutralize their anticoagulant activities.
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PMID:Neutralization of enoxaparine-induced bleeding by protamine sulfate. 216 53

The binding of plasma factor XIII to fibrinogen or fibrin that has been chemically or enzymatically induced to polymerize was studied. Factor XIII binding was assayed using a 3H-putrescine incorporation assay and an 125I-plasma factor XIII binding assay. More than 80% of the native and radiolabeled plasma factor XIII was bound to fibrin I formed by reptilase in EDTA, citrate, or heparin anticoagulated plasma. Plasma factor XIII and 125I-factor XIII was bound (89.6% to 92.5%) to fibrin II formed by thrombin in either citrate or EDTA anticoagulated plasma. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of 125I-plasma factor XIII bound to fibrin I or fibrin II formed by reptilase or thrombin in the presence of EDTA demonstrated the b2-subunit remained bound to the a-chains or thrombin-cleaved a-chains. In the presence of calcium chloride and thrombin, the b2-subunit dissociated and factor XIIIa was bound. Protamine sulfate caused fibrinogen polymerization in the absence of divalent cations and reduced both plasma factor XIII and immunologic fibrinogen levels. Fibrinogen polymerized by protamine sulfate bound plasma factor XIII and the a2-subunit of 125I-platelet factor XIII. Plasma factor XIII was also bound to sonicated non-cross-linked fibrin II in either normal plasma or afibrinogenemic plasma. Plasma levels of several coagulation proteins were unchanged after the addition of reptilase, protamine sulfate, or sonicated fibrin to plasma. These results demonstrate that a specific binding site for the a2-subunit of plasma factor XIII is present on polymerized fibrinogen, fibrin I, and fibrin II. Furthermore, the presence of divalent cations, thrombin-cleavage of plasma factor XIII, and release of fibrinopeptides A or B are not required for plasma factor XIII binding to polymerized fibrinogen and fibrin.
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PMID:Regulation of plasma factor XIII binding to fibrin in vitro. 241 26

The neutralization of the anticoagulant, anti-thrombin, and bleeding effects of dermatan sulfate (DS), a potential antithrombotic agent, was investigated. Protamine sulfate (PS) and hexadimethrine bromide (Polybrene), which reverse the anticoagulant effect of heparin, also neutralized DS in vitro. In human plasma, polybrene was approximately 3 times more active on a weight basis than PS for neutralizing DS (1.5 micrograms polybrene inhibits 1 microgram DS). Intravenous administration of polybrene to rabbits pretreated with DS in a 1:1 weight ratio immediately neutralized 90% of DS and this effect was stable with time. In contrast, PS in a weight ratio of 6:1 (PS to DS) only neutralized 50% of DS injected. When plasma DS concentrations were maintained by continuous infusion between 3 and 15 micrograms/ml, a bolus of polybrene 0.25 mg/kg induced an immediate drop of about 4 micrograms/ml but initial values of DS were recovered within 20 min. PS was again much less effective than polybrene for neutralizing DS. The bleeding effect of DS and its correction by polybrene was studied by using the rat tail transection model. Very large doses of DS (greater than 10 mg/kg) were required to get a modest prolongation of bleeding time. The injection of equivalent doses of polybrene in animals pretreated by DS induced a strong bleeding effect associated with a drop in platelet and leukocyte counts. Animal models are thus inappropriate for investigating the correction of DS-induced bleeding, because high doses of both DS and neutralizing agents are required in these models. Our results indicate that, provided the doses of neutralizing agents remain below their established levels of toxicity in man, DS could if necessary be neutralized completely by polybrene and partially by PS.
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PMID:Neutralization of dermatan sulfate in vitro and in vivo by protamine sulfate and polybrene. 272 57

Functional assays for heparin cofactor II (HC-II) are based on the inactivation of thrombin by HC-II in the presence of dermatan sulfate (DS). Residual thrombin is measured in a chromogenic assay. Interference by the antithrombin-III (AT-III)/heparin complex, which also rapidly inactivates thrombin, must be eliminated from the HC-II test system. Commercial DS is contaminated with heparin, while plasma specimens to be tested contain AT-III. After NaNO2/acetic acid treatment of DS (to inactivate heparin), there was enough residual heparin to cause AT-III interference. Treatment of plasma with commercially available anti-AT-III antiserum largely, but not completely, removed AT-III interference from the HC-II assay. With commercially available reagents, both NaNO2/acetic acid treatment of DS and anti-AT-III treatment of plasma were needed to eliminate heparin/AT-III interference. Protamine sulfate inactivated DS as well as heparin and could not be used to reduce AT-III/heparin interference with the HC-II assay.
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PMID:Heparin cofactor II assay. Elimination of heparin and antithrombin-III effects. 334 70

Protamine sulfate is considered a weak anticoagulant, yet little is known concerning the mechanism of this effect or its relation to prior heparin exposure. This investigation defined the influence of increasing doses of protamine, with and without prior heparin anticoagulation, on the activated clotting time (ACT), thrombin clotting time (TCT), prothrombin time (PT), partial thromboplastin time (PTT), fibrinogen level, platelet count, and platelet aggregation to ADP in dogs (n = 8). Four doses of intravenous protamine sulfate (1.5, 3.0, 6.0, and 15.0 mg/kg) were studied in each animal, with at least 5 days between individual studies. Four dogs received heparin, 150 IU/kg 10 min prior to protamine sulfate administration, and four dogs received protamine sulfate alone. Protamine sulfate caused anticoagulation, both in the presence and absence of heparin, with significant changes occurring in the ACT, PTT, platelet count, and platelet aggregation. Relevant changes did not occur in the TCT, PT, or fibrinogen levels. Platelet effects were capable of causing bleeding with standard or excess use of protamine sulfate, especially if platelet numbers were already decreased, as might occur in surgical procedures where thrombocytopenia commonly accompanies major blood loss and replacement. The ACT, reflecting both the coagulation cascade and platelet function, was the test most profoundly affected by protamine overdosage, and therefore may be misleading as a measure of protamine reversal of heparin. The TCT, which is sensitive to heparin anticoagulation but not protamine-induced anticoagulation, should be more accurate in differentiating inadequate heparin reversal from the effects of excess protamine.
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PMID:Anticoagulant effects of protamine sulfate in a canine model. 339 95

When commercially prepared porcine mucosal heparin is added to human plasma, some of the heparin fractions form a complex with antithrombin III activating it to an immediate inhibitor of thrombin as well as of other serine proteases. Certain fractions of heparin may complex with other proteins such as alpha 2-macroglobulin, another progressive inhibitor of thrombin. Without complexing with antithrombin III, this protein-bound heparin fraction(s) still retains the capacity to activate it to an immediate inhibitor of thrombin. Protamine sulfate inactivates those heparin fractions that bind to antithrombin III but not those bound to alpha 2-macroglobulin. Activated antithrombin III may undergo a molecular change in the presence of protamine which not only changes it back to a progressive inhibitor but makes it resistant to activation by the protein-bound heparin fraction(s). However, it can still be reactivated by other heparin fractions in fresh whole heparin. The observations presented may help explain heparin "rebound" in patients believed adequately neutralized with protamine.
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PMID:The interaction of protein-bound heparin and antithrombin III. 616 28

Protamine sulfate (salmine), a basic protein with a molecular weight of 4,626 +/- 109, is a known antiheparin agent which in the absence of heparin demonstrates an anticoagulant activity. To date, much work has been done to elucidate the interaction of heparin with thrombin and its physiologic inhibitor, Antithrombin III (ATIII). Little is known, however, about the mechanism of anticoagulant action of protamine sulfate and its mode of thrombin inactivation. We provide information about the interaction of protamine sulfate with purified, labeled thrombin and ATIII through binding experiments in which protamine is shown to inhibit the inactivation of thrombin by ATIII. Furthermore, we show in clotting assays that protamine sulfate has an inhibitory effect on thrombin in the conversion of fibrinogen to fibrin, and that this inhibition is concentration dependent, partial, and reversible.
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PMID:Interaction of protamine sulfate with thrombin. 684 26

Aprosulate or lactobionic acid is a highly sulfated analogue of heparin which is currently undergoing clinical trials in Europe as a potential antithrombotic drug. Aprosulate exerts a strong anticoagulant effect in plasma as a result of its interaction with heparin cofactor II. In this study, the ability of protamine sulfate to neutralize the anticoagulant activity of Aprosulate was investigated. In vitro, ex vivo, and in vivo coagulation studies were performed using various clotting assays such as the APTT, Heptest, and thrombin time as a measure of the anticoagulant activity of Aprosulate. In the first study, protamine sulfate when administered in vitro to plasma samples containing various concentrations of Aprosulate was found to effectively neutralize the anticoagulant activity of the Aprosulate in both normal human and normal monkey plasma systems. However, the relative index of neutralization of Aprosulate was assay dependent. Protamine sulfate was also found to antagonize the anticoagulant effects of Aprosulate in an ex vivo study. The ex vivo supplementation of protamine sulfate to plasma samples collected at various time intervals following the subcutaneous administration of Aprosulate to a group of primates completely neutralized the anticoagulant activity of the Aprosulate. In a third in vivo study, protamine sulfate when injected intravenously into the bloodstream of a group of primate was found to completely neutralize the anticoagulant effects of a previously administered dosage of Aprosulate. The results of these three studies clearly suggest that protamine sulfate can be used to effectively neutralize the anticoagulant activity of Aprosulate.
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PMID:Protamine sulfate neutralization of the anticoagulant activity of Aprosulate, a synthetic sulfated lactobionic acid amide. 801 19

Thrombin is inhibited by its cognate plasma inhibitor antithrombin, through the formation of covalent thrombin-antithrombin (TAT) complexes that are found as ternary complexes with vitronectin (VN-TAT). To determine whether the metabolism of VN-TAT ternary complexes is different from that previously reported for binary TAT complexes, plasma clearance studies were done in rabbits using human VN-TAT. 125I-VN-TAT was shown to be cleared rapidly from the circulation (t1/2alpha = 3.8 min) in a biphasic manner mainly by the liver. 125I-TAT had a similar initial clearance (t1/2alpha = 5.3 min) but had a significantly faster beta-phase clearance (t1/2beta = 42.8 min versus 85.4 min for VN-TAT; p = 0.005). Protamine sulfate and heparin abolished the rapid initial alpha-phase of 125I-VN-TAT clearance and reduced its liver-specific association and in vivo degradation. Heparin also reduced the alpha-phase clearance of 125I-TAT and was associated with the appearance of high molecular weight complexes, suggesting enhanced complex formation between VN and TAT. 125I-VN-TAT binding to HepG2 cells was reduced by competition with VN-TAT or heparin but to a much lesser extent in the presence of TAT. The binding of VN-TAT to HepG2 cells was not inhibited by competition with the low density lipoprotein receptor-related protein ligand, methylamine-alpha2-macroglobulin. 125I-VN-TAT binding was also inhibited by treating HepG2 cells with heparinase or by growing the cells in the presence of beta-D-xyloside. Finally, both heparin and chloroquine, but not methylamine-alpha2-macroglobulin, reduced the internalization and degradation of VN-TAT by HepG2 cells. Taken together, these data indicate the importance of VN in TAT metabolism and demonstrate that VN-TAT binds to liver-associated heparan sulfate proteoglycans, which mediate its internalization and subsequent intracellular degradation.
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PMID:In vivo clearance of ternary complexes of vitronectin-thrombin-antithrombin is mediated by hepatic heparan sulfate proteoglycans. 972 80

Acetaldehyde (AcH) at preincubation concentrations of 447, 89.4, and 17.9 mM potentiates the effects of heparin on the clotting time of plasma. While control plasma clotted in the range of 12.6+/-0.1 to 13.8+/-0.1 sec, and heparin-treated plasma clotted in a range from 131.5+/-2.5 to 168.2+/-1.2 sec, heparin that was preincubated at room temperature for 30 min with 89.4 or 447 mM AcH did not clot plasma in 300 sec. Heparin exposed to 17.9 mM AcH clotted plasma in 193+/-1.1 sec. Ethanol at a 404 mM concentration also prolonged the clotting time of heparin-treated plasma >300 sec, while 202 mM ethanol prolonged the clotting time of heparin-treated plasma from 149.0+/-2.0 sec to 219.5+/-1.7 sec. It is suggested that AcH alters the tertiary structure of heparin by adduct formation, possibly by formation of cyclic acetals with iduronic and glucuronic acids, thereby more readily affecting binding of the glycosaminoglycan to antithrombin III and/or thrombin, prolonging clotting time. Ethanol, which does not react covalently with heparin, might affect its conformation as a consequence of an organic solvent effect. Protamine sulfate prolonged the clotting time of plasma from 13.6+/-0.1 sec to 17.9+/-0.2 sec. Protamine sulfate-treated heparin clotted plasma in 21.0+/-0.4 sec relative to heparin-treated plasma (160.4+/-1.7 sec). In subsequent experiments, AcH-treated protamine sulfate extended the clotting time of protamine sulfate from 17.9+/-0 sec to 33.7+/-0.6 sec. Prior addition of protamine sulfate to AcH-heparin mixtures or heparin to protamine sulfate-AcH mixtures before addition to plasma resulted in clotting times of 22.0+/-0.4 sec and 24.1+/-0.5 sec, respectively, relative to control clotting times of 162.3+/-2.6 sec for plasma-heparin mixtures. These results confirm both the reduction in coagulation time of heparin-treated plasma by protamine sulfate and the prolongation of clotting time of plasma by protamine sulfate. Furthermore, and importantly, they indicate that acetaldehyde-treated protamine sulfate is a more effective anticoagulant than protamine sulfate. It is suggested that reversible adduct formation between acetaldehyde, heparin, and protamine sulfate may occur as a means explaining the essentially identical coagulation time of these mixtures when added to plasma regardless of the order of premixing. Ethanol (404 mM) did not influence protamine sulfate effects. Lastly, the potentiation of the anticoagulant function of heparin by acetaldehyde suggests that a structural modification of the glycosaminoglycan may occur in alcoholics.
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PMID:Coagulation protein function VI: augmentation of anticoagulant function by acetaldehyde-treated heparin. 1048 17


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