Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:4.2.2.7 (heparinase)
 1,270 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recombinant hirudin (r-hirudin) is currently under development as an anticoagulant for use in surgery, therapeutic anticoagulation, disseminated intravascular coagulation and other pathologic states involving the generation of thrombin. Circulating levels of r-hirudin as an antithrombotic agent range from 2 to 20 micrograms/ml (0.1-1.0 mg/kg) as determined in an animal model of stasis thrombosis. In order to establish a relationship between the r-hirudin circulating level and bleeding, we utilized a rabbit ear blood loss model. r-Hirudin did not produce any loss of blood at dosages up to 20 micrograms/ml i.v. (1.0 mg/kg). When the circulating levels were maintained at 20 micrograms/ml for periods of up to 3 h, no increase in blood loss was observed. At 50 and 100 micrograms/ml initial circulating levels (2.5 and 5.0 mg/kg) a dose-dependent increase in the blood loss was observed which was equivalent to that observed with 1.25 and 2.5 mg/kg i.v. heparin. Such levels of r-hirudin are not expected in clinical usage. In contrast to heparin, the anticoagulant actions of r-hirudin were not neutralized by protamine sulfate, platelet factor 4, other polycationic agents and heparinase. In our studies, the blood loss induced by greater than 2.0 mg/kg i.v. dosages of r-hirudin in an animal model was neutralized by the administration of an activated prothrombin complex concentrate at 25 U/kg. In a similar experimental setting, r-factor VIIa was also partially effective. These studies suggest that r-hirudin anticoagulation may not require neutralization, since bleeding effects are not observed at effective antithrombotic dosages in individuals with normal hemostatic status.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Some objective considerations for the neutralization of the anticoagulant actions of recombinant hirudin. 189 98

The structure of the glycosaminoglycan chain of a heparan sulfate proteoglycan isolated from the conditioned medium of an endothelial cell line has been analyzed by using various degradative enzymes (heparitinase I, heparitinase II, heparinase, glycuronidase, sulfatases) from Flavobacterium heparinum. This proteoglycan inhibits the thromboplastin-activated pathway of coagulation; as a consequence, the catalytic conversion of prothrombin to thrombin is arrested. Heparitinase I (EC 4.2.2.8), an enzyme with specificity restricted to the heparan sulfate portion of the polysaccharide, releases fragments with the electrophoretic mobility and the structure of heparin. Conversely, an assessment of the size and distribution of the heparan sulfate regions has been provided by the use of heparinase (EC 4.2.2.7), which, by degrading the heparin sections of the chain, releases two segments that exhibit the structure of heparan sulfate. One of these segments is attached to the protein core. On the basis of these findings, the heparan sulfate chain can be defined as a copolymer containing heparin regions in its structure. The combined use of these enzymes has made it possible to establish the disaccharide sequence of parts of the glycosaminoglycan moiety of this proteoglycan.
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PMID:Heparin sequences in the heparan sulfate chains of an endothelial cell proteoglycan. 295 57

The ascitic form of a chemically-induced pancreatic ductal adenocarcinoma in the Syrian golden hamster was very bloody and indistinguishable from blood macroscopically. Unlike blood, the bloody fluid remained unclotted at room temperature. To explore the possibility of presence of anticoagulants, we mixed 40% cell-free fluid with 60% normal human plasma and tested the clottability of the mixture with standard techniques. Plasma containing the fluid showed markedly prolonged activated partial thromboplastin time (APTT), thrombin time (TT) and recalcification time (RCT), and normal prothrombin time (PT) and reptilase time (RT). Comparing the prolongation of APTT of samples containing the fluid to those containing a commercial heparin, the fluid contained an anticoagulant activity equivalent to 0.436 +/- 0.03 unit heparin per ml (mean +/- SEM, n = 14). In addition to prolonging the APTT, TT and RCT, the fluid also inhibited the clotting and amidolytic activities of thrombin. "Heparsorb" had nearly completely neutralized the anticoagulant activity in fluid samples, while protamine sulfate was only partially effective. Incubation of fluid with pronase or phospholipase did not affect its anticoagulant activity; incubation with heparinase had only a minimal effect. Electrophoresis of an alkali digested fluid on cellulose acetate revealed the presence of heparan sulfate. The native ascitic fluid also contained other hemostatic components including platelets, fibrinogen and antithrombin III, but their concentrations were much lower than in blood. Apparently, heparan sulfate in the neoplastic effusion is largely responsible for the bloody ascites tumor remaining unclotted.
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PMID:Anticoagulant activity in cell-free peritoneal fluid of an experimental pancreatic ascites tumor. 300 55

An 8-month-old male with acute monoblastic leukemia died during induction chemotherapy of severe bleeding refractory to repeated infusions of platelets and clotting factors. A heparin effect was suggested by prothrombin time (PT) of 26 seconds, partial thromboplastin time (PTT) of 94 seconds, thrombin time 240 seconds, and reptilase time 18.4 seconds, with a fibrinogen of 88 mg/dl. Both plasma mixed with the patient's urine and the patient's plasma had their thrombin times corrected toward normal by both PF4 and protamine. Synergism of the anticoagulant with antithrombin III was demonstrated not only by enhanced inhibition of thrombin but also by an increased rate of formation of thrombin--antithrombin III complexes in the presence of the anticoagulant, which was eliminated by preincubation with heparinase. Since the anticoagulant activity was not found in the blasts themselves, it is presumed that the anticoagulant is heparin/heparan liberated from the endothelial lining by products of the cell destruction secondary to chemotherapy.
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PMID:A heparin-like anticoagulant in an 8-month-old boy with acute monoblastic leukemia. 658 79

A whole blood hemostasis system (Hepcon) provides both activated clotting time and accurate whole blood heparin concentration measurements via an automated protamine titration method. This study was designed to prospectively evaluate the impact of heparin and protamine administration using this system on the incidence and treatment of bleeding after cardiopulmonary bypass. Two hundred fifty-four patients requiring cardiopulmonary bypass were enrolled in this prospective study over a 7-month period. Patients treated with antifibrinolytic agents (aprotinin, epsilon-aminocaproic or tranexamic acid) were excluded. Patients were randomly assigned to either a control (n = 127) or intervention (n = 127) group. For control patients, the anticoagulation protocol consisted of an initial fixed dose of 250 U/kg of heparin, and additional 5000 U heparin doses were administered if the activated clotting time was less than 480 seconds. Heparin was neutralized with an initial fixed dose of protamine (0.8 mg protamine per milligram total heparin). For the intervention group, an initial dose of heparin was based on an automated heparin dose-response assay. Additional heparin doses were administered if the heparin concentration was less than the reference concentration or for an activated clotting time less than 480 seconds. The protamine dose was based on the residual heparin concentration. Treatment of excessive bleeding after cardiopulmonary bypass was based on an algorithm using point-of-care testing with whole blood prothrombin time, activated partial thromboplastin time, heparinase activated clotting time, and platelet count. No differences between the two treatment groups were identified in reference to demographic factors, preoperative anticoagulant medications, preoperative coagulation data, number of reoperations, or combined procedures and duration of cardiopulmonary bypass. Indirect evidence for coagulation factor consumption was demonstrated in control patients by more prolonged whole blood prothrombin time and activated partial thromboplastin time values after cardiopulmonary bypass when compared with values obtained in the intervention group. Patients in the intervention cohort received greater doses of heparin (intervention: 612 +/- 147, control: 462 +/- 114 U/kg, p < 0.0001) and had lower protamine to heparin ratios (intervention: 0.70 +/- 0.64, control: 0.94 +/- 0.21, p = 0.0001) compared with control patients. Patients in the intervention cohort received significantly fewer platelet (intervention: 1.7 +/- 3.6 U, control: 3.7 +/- 6.7 U, p = 0.003), plasma (intervention: 0.4 +/- 1.3 U, control: 1.4 +/- 2.5 U, p = 0.0001), and cryoprecipitate units (intervention: 0.0 +/- 0.0 U, control: 0.2 +/- 1.2 U, p = 0.04) during the perioperative interval than control patients.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The impact of heparin concentration and activated clotting time monitoring on blood conservation. A prospective, randomized evaluation in patients undergoing cardiac operation. 858 30

This study was designed to evaluate the potential in vitro use of heparinase to eliminate functionally active heparin prior to performing whole blood (WB) prothrombin time (PT) and activated partial thromboplastin time (APTT) assays. A total of 250 U/kg of heparin for cardiopulmonary bypass (CPB) was administered to 30 cardiac surgical patients in three consecutive, divided doses (20, 80, and 150 U/kg) at 15-min intervals. Blood specimens were obtained prior to heparin administration (baseline) and 10 min after each heparin dose. After collection, blood specimens were fractionated into three aliquots of which the first was used for determination of heparin concentration. After gentle mixing, WB PT and APTT measurements were performed for heparinase (Aliquot 2)- and nonheparinase (Aliquot 3)-treated blood. With consecutive heparin doses of 20 and 80 U/kg, WB PT increased from a baseline of 12.3 +/- 0.1 s to 13.3 +/- 0.2 and 18.5 +/- 1.3 s, while WB APTT increased from a baseline of 28.3 +/- 1.1 s to 89.5 +/- 5.4 after the initial heparin dose (20 U/kg). When compared to baseline (no heparin) results, small, progressive increases in heparinase-treated WB PT (0.7 +/- 0.1, 1.5 +/- 0.1, 2.1 +/- 0.1 s) and APTT (2.3 +/- 0.3, 5.7 +/- 0.4, 9.5 +/- 0.5 s) were seen with increasing heparin concentration (0.23, 1.58, and 3.95 U/mL, respectively). Heparinase was highly effective in eliminating the anticoagulant effects of even large amounts of heparin in plasma from cardiac surgical patients.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:In vitro reversal of heparin effect with heparinase: evaluation with whole blood prothrombin time and activated partial thromboplastin time in cardiac surgical patients. 794 73

The angiogenic factor, basic fibroblast growth factor (bFGF), is sequestered and protected by binding to heparan sulfate proteoglycans (HSPG) in the subendothelial extracellular matrix (ECM). Release of ECM-bound bFGF provides a novel mechanism for regulation of cell proliferation and neovascularization in normal and pathologic situations. Exposure of ECM to thrombin, the final activation product of the clotting cascade, resulted in release of high molecular weight HSPG-bFGF complex, as indicated by its immunoprecipitation with anti-bFGF antibodies, susceptibility to degradation by bacterial heparinase, and inhibition of its mitogenic activity in the presence of neutralizing anti-bFGF antibodies. The ECM-resident bFGF-HSPG complex was not released by thrombin in the presence of hirudin or antithrombin III, or by catalytically blocked thrombin preparations. A threefold to fivefold higher mitogenic activity was released by thrombin from ECM that was preheated (1 hour, 80 degrees C), as compared with native ECM. This difference is attributed to heat stable bFGF-HSPG complexes that are more readily released after heat treatment of the ECM and to activation and release of ECM-resident transforming growth factor-beta (TGF-beta) activity. Our results indicate that the large reservoir of proteolytic activity present in plasma in the form of prothrombin may participate in release from the subendothelial ECM of biologically active bFGF and TGF-beta, depending on the accessibility of thrombin. Thrombin may gain access to the subendothelium on clot formation after tissue injury and as a result of the conversion of prothrombin to thrombin induced by the ECM itself.
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PMID:Thrombin-induced release of active basic fibroblast growth factor-heparan sulfate complexes from subendothelial extracellular matrix. 850 69

In a multicentric study the influence of heparinase (Hepzyme) was evaluated on activated partial thromboplastin time, thrombin clotting time and prothrombin time using the recombinant human tissue factor and synthetic phospholipid (phosphatidylcholine and phosphatidyl-serine reagent). Hepzyme itself does not have any influence on normal coagulation values of activated partial thromboplastin time (aPTT) and prothrombin time (PT) assays whereas thrombin clotting time was prolonged by 10% (n = 60). In patients treated with unfractionated heparin for recent deep vein thrombosis (n = 47), plasma levels of aPTT, PT and thrombin clotting time (TCT) returned to the normal range in 100%, 97% and 91% after treatment with heparinase, respectively. Plasma samples of patients on coumarin were spiked with 2IU heparin/ml (n = 40) and were treated with heparinase. aPTT returned to baseline levels in 97.5%, PT in 99% and TCT in 69% of the samples. Plasma samples of patients receiving both heparin and coumarin were treated with heparinase (n = 18). aPTT and TCT values were shortened substantially and displayed the prolongation due to the effect of oral anticoagulants. PT values in these patients were also shortened. Freezing of plasma samples after treatment with heparinase resulted in a prolongation of the coagulation times in 15% of PT, 7% of aPTT and not of TCT values. The results show that treatment of plasma samples with heparinase abolishes the effect of unfractionated and low molecular weight heparin in vitro and ex vivo in patients during simultaneous treatment with oral anticoagulants. The use of heparinase may be of significance in patients with concomitant treatment of heparin and oral anticoagulants.
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PMID:Multicentric evaluation of heparinase on aPTT, thrombin clotting time and a new PT reagent based on recombinant human tissue factor. 883 97

We have previously demonstrated that thrombin possesses an active yet cryptic Arg-Gly-Asp (RGD) site which upon exposure induces endothelial cell (EC) adhesion via alpha nu beta 3 integrin [Bar-Shavit et al. (1991): J Cell Biol 112:335]. This was achieved in the presence of cell surface-associated heparan sulfate proteoglycans (HSPG) and exceedingly low concentrations of plasmin [Bar-Shavit et al. (1993): J Cell Biol 123:1279]. A portion of the cell surface-associated HSPG (glypican) is anchored via a covalently linked glycosyl-phosphatidylinositol (PI) residue, which can be released by treatment with glycosyl-PI-specific phospholipase C (PI-PLC). We report here that exposure of either bovine aortic EC, smooth muscle cells (SMC), or wild-type CHO cells to PI-PLC released HSPG involved in the conversion of thrombin to an adhesive molecule. The adhesion-promoting activity of the released HSPG was abolished following treatment with heparinase but not chondroitinase ABC. Incubation of thrombin with heparan sulfate-deficient CHO cells or cells that were pretreated with PI-PLC failed to induce its conversion to an adhesive molecule, indicating that glypican was playing a major role in this conversion. Moreover, affinity-purified glypican, but not syndecan or fibroglycan, elicited efficient conversion of plasmin-treated thrombin into an adhesive molecule. Antibodies raised against the RGD site in thrombin failed to interact with native thrombin, prothrombin, or the RGD site in other adhesive proteins such as vitronectin, fibrinogen, or fibronectin. Anti-thrombin-RGD antibodies which blocked the adhesion-promoting activity of thrombin were also capable of recognizing thrombin that was first incubated with a suboptimal concentration of plasm in in the presence of PI-PLC-released HSPG. Heparin, heparan sulfate, and PI-PLC-released HSPG had no effect on other cellular properties of thrombin such as receptor binding and growth-promoting activity. Altogether we have demonstrated that the heparin binding domain in thrombin plays a specific role in promoting thrombin adhesive properties and that membrane-associated glypican is likely to be the major physiological inducer of this property.
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PMID:Specific involvement of glypican in thrombin adhesive properties. 917 91

Heparin is usually obtained from mammalian organs, such as beef lung, beef mucosa, porcine mucosa, and sheep intestinal mucosa. Because of the increased use of heparin in the production of low-molecular-weight heparin (LMWH), there is a growing shortage of the raw material needed to produce LMWHs. A previous report described the structural features of a novel LMWH from the shrimp (Penaeus brasiliensis). In order to compare anticoagulant and antiprotease effects of this heparin, global anticoagulant tests, such as the prothrombin time, activated partial thromboplastin time, thrombin time, and Heptest, were used. Amidolytic anti-Xa and anti-IIa activities were also measured. The relative susceptibility of this heparin to flavobacterial heparinase was also evaluated. The United States Pharmacopeia (USP) potency of shrimp heparin (SH) was found to be 28 U/mg. SH produced a concentration-dependent prolongation of all of the clotting tests and exhibited marked inhibition of FXa and FIIa. Heparinase treatment resulted in a marked decrease of the anticoagulant effects and neutralized the in vitro anti-IIa actions. However, the anti-Xa activities were only partially neutralized. Protamine sulfate was only partially effective in neutralizing the anticoagulant and antithrombin effects of SH. SH also produced marked prolongation of activated clotting time, which was neutralized by heparinase but not by protamine sulfate. These results suggest that SH is a strong anticoagulant with comparable properties to mammalian heparins and can be used in the development of clinically useful antithrombotic-anticoagulant drugs.
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PMID:Anticoagulant and antiprotease effects of a novel heparinlike compound from shrimp (Penaeus brasiliensis) and its neutralization by heparinase I. 1119 Sep 4


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