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

It had been suggested that antithrombin activity on the surface of intact endothelial cells may play a role in inhibiting platelet adhesion and thrombus formation. The antithrombin activity may be due to thrombomodulin or to activation of antithrombin III by glycosaminoglycans or thrombomodulin, or possibly a combination of these. This inhibitory activity has been shown to be affected by such antiheparin agents as protamine, hexadimethrine bromide (Polybrene; Aldrich Chemical Co., Milwaukee, Wis.) and platelet factor 4, as well as by such enzymes as heparinase and heparitinase. We have used a hamster cheek pouch preparation to observe thrombus formation in vivo in a normal vascular flow, to determine whether the production of thrombi by thrombin can be enhanced by antiheparin agents. After intra-arterial injection or topical application of protamine or hexadimethrine bromide, platelet adhesion and thrombus formation on intact arteriolar endothelium was produced by a dose of thrombin, which when injected alone had no effect. No thrombi were found in venules or capillaries. Injection of heparin before or after the antiheparin agents necessitated a larger dose to enhance the action of thrombin. On electron microscopy the thrombi were found to consist primarily of platelets adherent to an intact endothelium. The possible clinical implications of these observations are discussed.
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PMID:Production of thrombi on intact endothelium by use of antiheparin agents in vivo. 224 59

In summary, the following points have been presented. PK 10169 produced somewhat weaker effects on the coagulant tests in comparison to heparin in various whole blood and citrated plasma assays. In the synthetic substrate assays, PK 10169 produced a pronounced inhibition of various serine proteases in the AT III supplemented system. No significant inhibition was noted in the non-AT III systems. Preliminary data show that PK 10169-AT III complex is capable of producing direct inhibition of the generation of factors Xa and XIIa. In all platelet function tests studied, this agent failed to produce any modulating effects. PK 10169 did not produce an effect on the fibrinolytic system in vitro. However, analysis of blood samples obtained from animals treated in vivo with this agent suggests activation of fibrinolysis. Thus, the mechanism of action must involve certain cellular components or endogenous modulation of the heparin fraction. The newly developed FPA generation test can be modified by various activators or blood systems to mimic closely in vivo physiology. PK 10169 produces a dose response that is more sensitive and more global by this method than the amidolytic anti-Xa or anti-IIa. In contrast to heparin, larger amounts of platelet factor 4 and protamine sulfate are needed to neutralize the anti-Xa and anti-IIa actions of this agent. Additionally, the anti-IIa component is more susceptible to neutralization than the anti-Xa component. Our studies also suggest that PK 10169 is resistant to the action of certain heparin digestive systems, such as heparinase.
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PMID:In vitro coagulant and amidolytic methods for evaluating the activity of heparin and a low molecular weight derivative (PK 10169). 388 97

Bovine antithrombin III (AT III) interaction with the luminal surface of bovine aortic segments with a continuous layer of endothelium was examined. Incubation of 125I-AT III with vessel segments, previously washed free of endogenous AT III, demonstrated specific, time-dependent binding to the protease inhibitor to the endothelium. Half-maximal binding was observed at an added AT III concentration of 14 nM. Binding of 125I-AT III to the vessel wall was reversible (50% dissociated in 4 min), and addition of either heparin or Factor Xa accelerated displacement of 125I-AT III from the vessel segment. Dissociation of 125I-AT III from the vessel segment in the presence of factor Xa coincided with the formation of a Factor Xa-125I-AT III complex. Inactivation of Factor IXa and Factor Xa by AT III was facilitated in the presence of vessel segments. Pretreatment of vessel segments with highly purified Flavobacterium heparinase precluded the vessel-dependent augmentation of AT III anticoagulant activity as well as specific binding of 125I-AT III to the vessel endothelium. In contrast, pretreatment of the vessel segments with chrondroitinases (ABC or AC) had no detectable effect on 125I-AT III binding or on AT III anticoagulant activity. AT III binding to vessel segments was competitively inhibited by increasing concentration of platelet factor 4. Binding of the protease inhibitor to vessel segments was inhibited by chemical modification of AT III lysyl or tryptophan residues. These AT III derivatives retained progressive inhibitory activity. These data suggest that heparin-like molecules are present on the aortic vessel wall and mediate binding of AT III to the vessel surface, as well as enhancing the anticoagulant activity of AT III at these sites.
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PMID:Interaction of antithrombin III with bovine aortic segments. Role of heparin in binding and enhanced anticoagulant activity. 396 7

Proteins with affinities for specific glycosaminoglycans (GAC's) were used as probes for testing the potential of cell surface GAG's to mediate cell adhesive responses to extracellular matrices (ECM). Plasma fibronectin (FN) and proteins that bind hyaluronate (cartilage proteo-glycan core and link proteins) or heparan sulfate (platelet factor 4 [PF4]) were adsorbed to inert substrata to evaluate attachment and spreading of several 3T3 cell lines. Cells failed to attach to hyaluronate-binding substrata. The rates of attachment on PF4 were identical to those on FN; however, PF4 stimulated formation of broad convex lamellae but not tapered cell processes fibers during the spreading response. PF4-mediated responses were blocked by treating the PF4-adsorbed substratum with heparin (but not chondroitin sulfate), or alternatively the cells with Flavobacter heparinum heparinase (but not chondroitinase ABC). Heparinase treatment did not inhibit cell attachment to FN but did inhibit spreading. Cells spread on PF4 or FN contained similar Ca2+-independent cell-substratum adhesions, as revealed by EGTA-mediated retraction of their substratum-bound processes. Microtubular networks reorganized in cells on PF4 but failed to extend into the broadly spread lamellae, where fine microfilament bundles had developed. Stress fibers, common on FN, failed to develop on PF4. These experiments indicate that (a) heparan sulfate proteoglycans are critical mediators of cell adhesion and heparan sulfate-dependent adhesion via PF4 is comparable in some, but not all, ways to FN-mediated adhesion, (b) the uncharacterized and heparan sulfate-independent "cell surface" receptor for FN permits some but not all aspects of adhesion, and (c) physiologically compatible and complete adhesion of fibroblasts requires binding of extracellular matrix FN to both the unidentified "cell surface" receptor and heparan sulfate proteoglycans.
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PMID:Cell surface heparan sulfate mediates some adhesive responses to glycosaminoglycan-binding matrices, including fibronectin. 621 15

We have previously reported that platelet factor 4 (PF4) inhibits human erythroleukemic (HEL) cell growth in a dose-dependent fashion in vitro and that PF4 binds to HEL cells in a specific, saturable, and concentration-dependent manner. In this article we demonstrate that the binding of PF4 on HEL cells and its inhibitory effect on HEL cell growth were mediated by heparan sulfate. We found that binding of iodine 125-labeled PF4 to HEL cells was inhibited by heparin, heparan sulfate, and dermatan sulfate and to a smaller extent by chondroitin sulfate. Ninety percent of 125I-labeled PF4 bound to HEL cells was released by cells after exposure to heparin and heparan sulfate. Treatment of cells with heparitinase and heparinase induced a decrease in the binding of 125I-labeled PF4 to cells. Binding of 125I-labeled PF4 was partially inhibited by the presence of increasing concentrations of protamine sulfate and basic fibroblast growth factor. To test whether PF4 bound to cell surface proteoglycans, proteoglycan synthesis was inhibited by using 4-methylumbelliferyl-beta-D-xyloside. The binding of 125I-labeled PF4 on treated cells was decreased, and xyloside treatment of cells abrogated the biologic activity of PF4 in a plasma clot culture system. The inhibitory effect of PF4 was retained in a serum-free agar culture system, which indicates that the binding of PF4 to HEL cells induces cell growth inhibition in a direct fashion. Taken together, these findings suggest that PF4 directly acts on HEL cell growth by fixation on heparan sulfate proteoglycans on the HEL cell surface.
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PMID:Inhibitory effect of platelet factor 4 on human erythroleukemic cells is dependent on cell surface heparan sulfate. 865 41

Foot-and-mouth disease virus (FMDV) enters cells by attaching to cellular receptor molecules of the integrin family, one of which has been identified as the RGD-binding integrin alpha(v)beta3. Here we report that, in addition to an integrin binding site, type O strains of FMDV share with natural ligands of alpha(v)beta3 (i.e., vitronectin and fibronectin) a specific affinity for heparin and that binding to the cellular form of this sulfated glycan, heparan sulfate, is required for efficient infection of cells in culture. Binding of the virus to paraformaldehyde-fixed cells was powerfully inhibited by agents such as heparin, that compete with heparan sulfate or by agents that compete for heparan sulfate (platelet factor 4) or that inactivate it (heparinase). Neither chondroitin sulfate, a structurally related component of the extracellular matrix, nor dextran sulfate appreciably inhibited binding. The functional importance of heparan sulfate binding was demonstrated by the facts that (i) infection of live cells by FMDV could also be blocked specifically by heparin, albeit at a much higher concentration of inhibitor; (ii) pretreatment of cells with heparinase reduced the number of plaques formed compared with that for untreated cells; and (iii) mutant cell lines deficient in heparan sulfate expression were unable to support plaque formation by FMDV, even though they remained equally susceptible to another picornavirus, bovine enterovirus. The results show that entry of type O FMDV into cells is a complex process and suggest that the initial contact with the cell surface is made through heparan sulfate.
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PMID:Efficient infection of cells in culture by type O foot-and-mouth disease virus requires binding to cell surface heparan sulfate. 876 38

The mechanisms by which the intracellular pathogen Listeria monocytogenes interacts with the host cell surface remain largely unknown. In this study, we investigated the role of heparan sulfate proteoglycans (HSPG) in listerial infection. Pretreatment of bacteria with heparin or heparan sulfate (HS), but not with other glycosaminoglycans, inhibited attachment and subsequent uptake by IC-21 murine macrophages and CHO epithelial-like cells. Specific removal of HS from target cells with heparinase III significantly impaired listerial adhesion and invasion. Mutant CHO cells deficient in HS synthesis bound and internalized significantly fewer bacteria than wild-type cells did. Pretreatment of target cells with the HS-binding proteins fibronectin and platelet factor 4, or with heparinase III, impaired listerial infectivity only in those cells expressing HS. Moreover, a synthetic peptide corresponding to the HS-binding ligand in Plasmodium falciparum circumsporozoite protein (pepPf1) inhibited listerial attachment to IC-21 and CHO cells. A motif very similar to the HS-binding site of pepPf1 was found in the N-terminal region of ActA, the L. monocytogenes surface protein responsible for actin-based bacterial motility and cell-to-cell spread. In the same region of ActA, several clusters of positively charged amino acids which could function as HS-binding domains were identified. An ActA-deficient mutant was significantly impaired in attachment and entry due to altered HS recognition functions. This work shows that specific interaction with an HSPG receptor present on the surface of both professional and nonprofessional phagocytes is involved in L. monocytogenes cytoadhesion and invasion and strongly suggests that the bacterial surface protein ActA may be a ligand mediating HSPG receptor recognition.
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PMID:Host cell heparan sulfate proteoglycans mediate attachment and entry of Listeria monocytogenes, and the listerial surface protein ActA is involved in heparan sulfate receptor recognition. 897 95

Heparin, the most widely used antithrombin, suffers several limitations, including high inter-individual variability of anticoagulant response, a nonlinear dose-response curve, inability to inactivate clot-bound thrombin, a requirement for endogenous cofactors and inactivation by platelet factor 4 and heparinase. These shortcomings may explain its suboptimal efficacy and safety in the prevention of arterial vessel occlusion. Heparin's drawbacks may be overcome by direct thrombin inhibitors. The development of these specific antithrombins has been a major therapeutic goal of the past decade. The high expectations generated by the use of these compounds in experimental models of arterial thrombosis appeared to be confirmed by the initial phase I and II clinical studies. However, large phase III trials have been highly discouraging: three trials with hirudin have been interrupted as a result of a high incidence of serious adverse events. Two of these trials were subsequently restarted at lower doses and did not support an incremental efficacy of hirudin over heparin. Two trials in the setting of angioplasty (one with hirudin and one with hirulog) have also failed to demonstrate the superiority of these compounds over heparin. Is this the result of a very narrow therapeutic range of these agents or the consequence of poor design of the phase II studies leading to the selection of inappropriate doses for the comparative efficacy trials? This review focuses on the clinical development of two specific antithrombins: hirudin and hirulog. The experimental pharmacology and human studies of Argatroban are discussed in a review by Fitzgerald and Murphy.
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PMID:Direct thrombin inhibitors in cardiovascular disease. 921 Oct 51

Platelet factor 4 is a tetrameric heparin binding chemokine released from the alpha-granules of activated platelets. In this study we show that platelet factor 4 binds with high affinity and specificity to an approximately 9-kDa sequence in heparan sulfate, which it protects from degradation by heparinase enzymes. This protected fragment is enriched in N-sulfated disaccharides and iduronate 2-O-sulfate residues, the latter being important for binding to platelet factor 4. The major structural motif of the fragment appears to consist of a pair of sulfated domains positioned at both ends separated by a central mainly N-acetylated region. On the basis of these findings, we propose a model in which the heparan sulfate fragment wraps around the ring of positive charges on platelet factor 4 with the iduronate 2-O-sulfates within the sulfated domains binding strongly to lysine clusters on opposite faces of the tetramer.
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PMID:Specific binding of the chemokine platelet factor 4 to heparan sulfate. 925 63


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