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Biomedical subjects

M R Buchanan

Publications and source records attributed to M R Buchanan.

At least 37 records · Page 2Linked to original sources
PubMed1994

Prevention of thrombus formation and growth by antithrombin III and heparin cofactor II-dependent thrombin inhibitors: importance of heparin cofactor II.

Heparin (HEP) prevents thrombus formation (TF) and thrombus growth (TG), by accelerating thrombin (THR) inhibition by antithrombin III (ATIII). Recent studies suggest that dermatan sulphate which catalyzes thrombin inhibition by heparin cofactor II (HCII), can inhibit TF and TG as effectively as HEP. This study compared the antithrombotic effects of HEP and another agent, Sulodexide (SLX) which catalyzes thrombin inhibition by ATIII and HCII simultaneously. TF was induced in rabbit jugular veins, using the stasis/hypercoagulation model. TG was measured as the accretion of 125I-fibrin onto existing thrombi in rabbit jugular veins. HEP and SLX inhibited TF when given in doses of 10 and 5 anti-thrombin U/kg, respectively. SLX (16 anti-thrombin U/kg or 260 micrograms/kg) was more effective than HEP (120 anti-thrombin U/kg or 800 micrograms/kg) in preventing TG when administered either as a bolus or by continuous infusion. These data suggest that agents which accelerate THR inhibition by both ATIII and HCII simultaneously, can inhibit TF and TG with less systemic anticoagulation than comparable antithrombotic doses of HEP.

Animals↗
PubMed1994

Influence of endothelium-derived relaxing factor on platelet function and hemostasis in vivo.

Endothelium-derived relaxing factor (EDRF) is a potent vasodilator, and is also, in vitro, a platelet-inhibitor. Experiments were performed to determine whether systemically released EDRF inhibits platelet-dependent hemostasis in vivo. Rabbits were treated with agents to release or block EDRF, and 5 standardized incisions were made in the ear. Carbachol, infused to stimulate EDRF release, abruptly lowered the blood pressure and caused increased bleeding. Neither effect was attributable to prostacyclin since neither was blocked by treatment of the rabbits with acetylsalicylic acid. In contrast, both the hypotension and bleeding were attenuated by the selective antagonist of EDRF synthesis, NG-nitro-L-arginine. However, neither the hypotension nor the bleeding associated with carbachol was inhibited by an infusion of free hemoglobin, used to scavenge intraluminally-released EDRF. We conclude that in this model endogenously-released EDRF increases bleeding indirectly by provoking vasodilatation, rather than directly by inhibiting platelet function.

Animals↗
PubMed1994

The relative roles of vitronectin receptor, E-selectin and alpha 4 beta 1 in cancer cell adhesion to interleukin-1-treated endothelial cells.

Adhesion of cancer cells to endothelium is thought to be a prerequisite to extravasation during the haematogenous phase of metastasis, and is enhanced after perturbation of the endothelium by interleukin-1 (IL-1). The inducible endothelial adhesion molecules, E-selectin, VCAM-1/alpha 4 beta 1 and vitronectin receptor have been reported to mediate attachment of cancer cells to IL-1-treated endothelial cells. We have examined the relative contribution of these molecules by quantifying the adhesion of a panel of 22 human, 125I-labelled cancer cells and the rat W256 tumour to untreated and IL-1-treated endothelial monolayers in the presence of relevant neutralising antibodies. Antibodies against E-selectin inhibited the adhesion of HL-60 leukaemia cells and two colon carcinomas. Anti-alpha 4 beta 1 antibodies blocked adhesion of four melanomas, five sarcomas and one lung carcinoma. Anti-vitronectin receptor antibodies inhibited adhesion of 14 of the 22 human cell lines to IL-1-treated endothelial cells. Adhesion of seven cell lines was inhibited by more than a single antibody. In contrast, adhesion of one of the cancer cell lines was unaffected by any of the antibodies, suggesting involvement of other IL-1-inducible endothelial adhesion molecules. Moreover, none of the antibodies altered the attachment of cancer cells to unstimulated endothelial monolayers. We conclude that the mechanisms of cancer cell adhesion to the endothelium are influenced by endothelial activation and by the adhesive repertoire of the cancer cell.

Animals↗
PubMed1994

Is heparin the ideal anticoagulant for cardiopulmonary bypass? Dermatan sulphate may be an alternate choice.

Performance of cardiopulmonary bypass (CPB) during cardiac surgery requires the administration of high dose heparin to prevent CPB pump occlusion. However, this heparin use is associated with bleeding side-effects. Moreover, at the end of CPB, the heparin must be neutralized with protamine sulphate, which is also associated with adverse side-effects. A number of recent studies suggest that dermatan sulphate may be useful as an alternate anticoagulant to heparin. We determined whether CPB could be performed using dermatan sulphate instead of heparin, in an adult pig CPB model. When heparin was used, a high dose (> 200 U/kg, which generated > 3 anti-thrombin U/ml of plasma), was required to perform successful CPB and to maintain CPB pump patency. This dose was associated with a post CPB bleeding of approximately 600 ml/2 h. In contrast, successful CPB could be achieved when the pigs were given lower doses of dermatan sulphate than heparin, which in turn, were associated with less bleeding. We conclude that dermatan sulphate may be an alternate anticoagulant for cardiac surgery.

Animals↗
PubMed1993

Thrombin generation during cardiac surgery: is heparin the ideal anticoagulant?

Blood samples were collected from 43 patients undergoing elective cardiac surgery to determine the extent of thrombin generation and inhibition in patients when receiving heparin while undergoing cardiopulmonary bypass (CPB). Plasma prothrombin fragment F1 + 2 and thrombin-antithrombin III (TAT) levels were measured as markers of thrombin generation and inhibition, respectively. Both F1 + 2 and TAT levels increased significantly during the course of CPB despite the heparin causing significant systemic anticoagulation, i.e. the activated coagulation time (ACT) was prolonged to greater than 400 s throughout the entire surgical procedure. The extent of thrombin generation increased with time on CPB but did not differ between patients receiving normothermic and hypothermic cardioplegia during CPB. Furthermore, thrombin generation increased following the neutralization of the heparin with protamine sulphate, and continued to be elevated significantly 24 h post surgery. The observation that high dose heparin did not prevent thrombin generation during CPB, is consistent with previous experimental studies demonstrating that thrombin bound to fibrin or other surfaces (e.g. the CPB conduit) is resistant to antithrombin III/heparin inhibition, and thus able to facilitate further thrombin generation. The observation that thrombin generation continued to be elevated post surgery i.e. 24 h after neutralizing the heparin with protamine sulphate, suggests that the high dose heparin did not inhibit effectively all of the thrombin that had been generated. Thus, CPB patients may be at risk not only of bleeding and other side-effects associated with the acute use of high dose heparin, but may also be at risk of further thrombosis-related events either acutely or chronically.

Aged↗
PubMed1993

Localization of 13-hydroxyoctadecadienoic acid and the vitronectin receptor in human endothelial cells and endothelial cell/platelet interactions in vitro.

Blood/vessel wall cell interactions depend, in part, on the expression of adhesion receptors on cell surfaces, such as expression of the vitronectin receptor (VnR) on the apical surface of endothelial cells (ECs) for platelet/EC adhesion. However, it is unclear how receptor expression is regulated from within cells. In previous studies, we found that ECs metabolize linoleic acid into the lipoxygenase monohydroxide, 13-hydroxyoctadecadienoic acid (13-HODE), and that the intracellular level of 13-HODE correlates inversely with VnR expression and platelet adhesion to the EC apical surface. In this study, we determined the physical associations of 13-HODE and VnR in unstimulated and stimulated ECs, ie, at times when ECs were and were not adhesive for specific ligands and platelets, using double antibody immunofluorescent staining techniques and binding assays. 13-HODE and the VnR were colocalized within unstimulated ECs. When ECs were stimulated, 13-HODE was no longer detectable, either in or outside the ECs, and the VnR was detected on the apical surface of the ECs. These changes were paralleled by increased vitronectin binding and increased platelet adhesion to the ECs. We suggest that colocalization of 13-HODE with VnR reflects a 13-HODE/VnR interaction, confining the VnR in a nonadhesive form inside unstimulated ECs, and, as a result, the ECs are nonadhesive. When the ECs are stimulated, 13-HODE and VnR dissociate, allowing the VnR to relocate on the EC surface, where the VnR undergoes a conformational change resulting in increased EC adhesivity.

Blood Platelets↗
PubMed1993

The effects of heparin and annexin V on fibrin accretion after injury in the jugular veins of rabbits.

We compared the relative abilities of unfractionated heparin and annexin V to prevent fibrin accretion onto injured jugular veins in vivo. Heparin was used to accelerate the inhibition of thrombin by antithrombin III, and annexin V was used to inhibit the assembly of the prothrombinase complex on phospholipid surfaces, thereby blocking thrombin generation. Rabbit jugular veins were isolated in situ, a 2 cm segment was injured by perfusing it with air, and then blood flow was re-established. Five minutes later, each rabbit was injected with heparin (20 U/kg) or annexin V (0.3 mg/kg) and then with 125I-fibrinogen. The amount of 125I-fibrin accumulation onto each injured vessel wall segment was measured 4 h later. Each injured vessel was completely de-endothelialized as a result of the air perfusion as demonstrated by electron microscopy. 125I-fibrin accretion onto the injured jugular veins was enhanced 2.4-fold as compared to the uninjured veins in sham-operated animals. Heparin treatment did not reduce fibrin accretion, whereas, annexin V treatment decreased fibrin accretion by 60%, p < 0.05. This latter effect was achieved without sustained circulating anticoagulation. Additional experiments confirmed that the inhibitory effect of annexin V on fibrin accretion was associated with a surface specific effect, since more annexin V bound to the injured jugular vein segments as compared to the non-injured jugular veins.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Interleukin 1-induced cancer cell/endothelial cell adhesion in vitro and its relationship to metastasis in vivo: role of vessel wall 13-HODE synthesis and integrin expression.

Previously, we have demonstrated that stimulation of endothelial cells (ECs) with interleukin-1 alpha (IL-1 alpha) enhances the synthesis and expression of the vitronectin receptor (VnR), promotes VnR-dependent adhesion of human A549 adenocarcinoma cells to ECs, and is associated with decreased EC 13-hydroxyoctadecadienoic acid (13-HODE) synthesis in vitro. To determine whether these observations are relevant in vivo, we examined the acute retention and subsequent metastasis of intravenously-injected B16F10 melanoma cells in murine lungs, in relation to vessel wall 13-HODE. In C57BL/6 mice pretreated with IL-1 alpha, vessel wall 13-HODE was decreased and B16F10 lung entrapment and metastasis were increased. The latter two events were blocked by pretreating the animals with the GRGDS peptide. These data suggest a relationship between vessel wall 13-HODE synthesis, adhesion molecule expression, and adhesion of B16F10 cells to the endothelium.

Animals↗
PubMed1993

Platelet adhesivity to subendothelium is influenced by polymorphonuclear leukocytes: studies with aspirin and salicylate.

Interactions between blood platelets and polymorphonuclear leukocytes (PMN) influence cell reactivity. We have examined the effects of inhibiting platelet and PMN cyclooxygenase and lipoxygenase pathways, by treatment with aspirin (ASA) and salicylate (SAL) on platelet-vessel wall interaction studied under flow conditions by means of an annular chamber perfusion system. We have also measured the levels of cyclooxygenase-derived metabolites during perfusion. Perfusates were prepared with untreated and ASA- or SAL-treated PMN or platelets. Our results demonstrated that blockage of the lipoxygenase pathway in PMN significantly increased platelet thrombus formation and favored the production of thromboxane B2 (TxB2) during perfusion, whereas inhibition of cyclooxygenase pathway in PMN had no effect either on platelet deposition or on TxB2 levels. In contrast, blockage of platelet cyclooxygenase, which caused almost total inhibition of TxB2, enhanced platelet adhesion and did not modify platelet thrombus formation. These results suggest that under dynamic conditions cooperative metabolic mechanisms between platelets and PMN directly influence platelet interaction with vascular subendothelium.

6-Ketoprostaglandin F1 alpha↗
PubMed1993

Prevention and treatment of thrombosis: novel strategies arising from our understanding the healthy endothelium.

The strategies used to prevent and treat thrombosis are based on our understanding of how to ameliorate the pathophysiological responses to injury, such as using inhibitors of platelet activation and inhibitors of coagulation. Both platelets and the coagulation pathway are activated in response to injury. An alternative strategy which has not been investigated to the same extent, is to use substances which contribute to the biocompatibility of blood and vessel wall which predominate under normal conditions. This latter strategy exploits the concept of restoring blood/vessel wall compatibility without risk of bleeding such as can occur when platelets as rendered haemostatically defective (antiplatelet therapies) or maintaining the patient hypocoagulated (anticoagulant therapies). Recent studies suggest that by better understanding the biocompatibility of the healthy endothelium with blood, we may be able to identify those substances which contribute to blood/vessel wall biocompatibility in the healthy environment, and subsequently which may achieve effective antithrombotic therapy with minimal risk of bleeding side-effects. In this review, we identify three such substances all of which are produced by the blood vessel wall. These agents are 13-hydroxyoctadecadienoic acid (13-HODE), a lipoxygenase metabolite of linoleic acid, and two glycosaminoglycans, heparan sulfate and dermatan sulfate.

Animals↗
PubMed1992

Prevention of thrombus growth by antithrombin III-dependent and two direct thrombin inhibitors in rabbits: implications for antithrombotic therapy.

We compared the abilities of heparin and two direct thrombin inhibitors to prevent fibrin accretion onto pre-existing thrombi in rabbits. Inhibition of thrombus growth was measured as the ability of each test compound to inhibit the accretion of 125I-fibrin onto thrombi pre-formed in jugular veins of rabbits. When administered as a continuous infusion, the two direct (i.e. antithrombin III-independent) thrombin inhibitors, r-hirudin and a tripeptide, Ac(D)-Phe-Pro-bor-Arg (P-8714) inhibited fibrin accretion as effectively as heparin, but did so in doses which generated little systemic anticoagulation, as compared to the marked anticoagulation associated with the heparin effect. However, both r-hirudin and P-8714 were more effective when they were administered as a single bolus injection than as a continuous infusion. Under the former conditions, there was only a transient systemic anticoagulant effect. We conclude that direct or antithrombin III-independent thrombin inhibitors are more effective than heparin in preventing thrombus growth. The limited effect of heparin is likely due to fibrin impairing the ability of heparin/antithrombin III to inactivate thrombin.

Amino Acid Sequence↗
PubMed1992

Inhibition of the amplification reactions of blood coagulation by site-specific inhibitors of alpha-thrombin.

Hirudin and hirulog-1 [D-Phe-Pro-Arg-Pro-[Gly]4-desulphohirudin-(54-65)] abrogate the enzyme activities of alpha-thrombin by binding the enzyme simultaneously at its catalytic centre and fibrin(ogen)-recognition exosite. In contrast, hirugen [hirudin-(54-65)] binds alpha-thrombin solely at the fibrin(ogen)-recognition exosite, and competitively inhibits fibrinopeptide A release. To investigate the extent to which the fibrin(ogen)-recognition exosite is involved when alpha-thrombin catalyses the amplification reactions of coagulation, we compared the abilities of hirudin, hirulog-1 and hirugen to inhibit simultaneously Factor X, Factor V and prothrombin activation. Whereas 0.1 microM-hirudin and 0.1 microM-hirulog-1 (i.e. less than 10% of the concentration of prothrombin in plasma) inhibited Factor X, Factor V and prothrombin activation, 10 microM was the minimum concentration of hirugen to achieve a similar anticoagulant action. Concentrations of hirudin and hirulog-1 equimolar to and 5 times greater than those of alpha-thrombin respectively abrogated Factor V activation by exogenous alpha-thrombin. In contrast, a 500-fold molar excess of hirugen could not. The inability of hirugen to inhibit the activation of the three clotting factors effectively suggests that the fibrin(ogen)-recognition exosite does not play a mandatory role when thrombin activates Factor V.

Amino Acid Sequence↗
PubMed1992

Up-regulated biosynthesis and expression of endothelial cell vitronectin receptor enhances cancer cell adhesion.

Extravasation of circulating cancer cells during metastasis is thought to involve adhesion to the vascular endothelium. To characterize this process, we measured the attachment of A549 human lung carcinoma cells to monolayers of cultured human umbilical vein endothelial cells. Pretreatment of the endothelial cells with 10 ng/ml interleukin 1 alpha (IL-1) for 4 h increased cancer cell attachment 2-5-fold. This increase was blocked by 100 microM glycyl-arginyl-glycyl-aspartyl-serine peptide and was decreased 60 +/- 10% (SD) by a vitronectin receptor polyclonal antiserum or 56 +/- 8% by a vitronectin receptor monoclonal antibody, LM609. Glycyl-arginyl-glycyl-aspartyl-serine or the vitronectin receptor antibodies did not inhibit cancer cell attachment to untreated endothelial cells. A fibronectin receptor antiserum had no effect on attachment to untreated or IL-1-treated endothelial cells. Pretreatment of endothelial cells with IL-1 increased their adhesion to fibronectin and vitronectin and increased the expression of vitronectin receptor and fibronectin receptor as detected by immunofluorescence flow cytometry, quantitative antibody binding, and immunoprecipitation of [35S]methionine-labeled cell extracts. IL-1 pretreatment also increased beta 1, beta 3, and alpha, integrin mRNA. The A549 cells did not express vitronectin receptor, since LM609 did not inhibit A549 adhesion to vitronectin or bind to A549 cells in flow cytometry, and vitronectin receptor antisera failed to immunoprecipitate vitronectin receptor from A549 cells. Furthermore, the beta 3 complementary DNA probe failed to hybridize to A549 RNA. A549 cells did express fibronectin receptor, which was increased by IL-1 treatment. We conclude that IL-1 induces the expression of both vitronectin receptor and fibronectin receptor on endothelial cells and that vitronectin receptor, in turn, facilitates A549 cell adhesion to endothelial cells.

Antibodies↗
PubMed1992

Eicosanoids, other fatty acid metabolites and the cardiovascular system: are the present antithrombotic approaches rational?

Antiplatelet +/- anticoagulant drugs are currently used as the standard treatment to prevent and treat thrombosis. While this approach is beneficial, it is not optimal. Recent evidence suggests that constituents of the vascular endothelium play an important role in regulating vessel wall thrombogenecity, thereby inhibiting thrombogenesis. These include constituents such as PGI2, tissue plasminogen activator, thrombomodulin and the lipoxygenase fatty acid metabolite derived from linoleic acid, 13-hydroxyoctadecadienoic acid (13-HODE). Consequently, new strategies have been developed to maximize the use of these agents for antithrombotic therapy. We will review these different approaches, discuss their rationale, and based upon recent experimental data, introduce an alternative approach for antithrombotic therapy which may circumvent a number of limitations and side-effect of the currently used drugs.

Animals↗
PubMed1991

Comparative effects of heparin and LMW heparin on hemostasis.

We performed studies to investigate the effect of protamine sulfate neutralization on both the anticoagulant and hemostatic effects of enoxaparin and heparin. Although the anti-Factor Xa effect of enoxaparin was incompletely neutralized by protamine sulfate in an experimental animal model, protamine sulfate reversed bleeding induced by hemorrhagic doses of enoxaparin.

Animals↗
PubMed1991

Endothelial cell damage by Walker carcinosarcoma cells is dependent on vitronectin receptor-mediated tumor cell adhesion.

The transport of cancer cells from blood vessels to extravascular tissue is a critical step in metastasis, where endothelial cells and the vascular basement membrane act as barriers to cell traffic. Because endothelial injury can facilitate the metastasis of intravascular cancer cells in vivo, the authors have studied in vitro the free-radical-mediated endothelial damage caused by the rat Walker 256 carcinosarcoma (W256) cell after stimulation with 10(-6) mol/l (molar) phorbol ester. Here the authors have examined the hypothesis that W256 cell-mediated endothelial injury is dependent on adhesion between the effector and target cells. Attachment of phorbol 12-myristate, 13-acetate (PMA)-stimulated W256 cells to endothelial monolayers was increased 1.8 +/- 0.1-fold and damage (3H-2-deoxyglucose release from labeled endothelium) 1.4 +/- 0.1-fold after 4-hour pretreatment of the endothelium with 10 ng/ml recombinant human interleukin-1 alpha (rIL-1 alpha). Under various assay conditions, the release of 3H-2-deoxyglucose correlated directly with tumor cell adhesion (r = 0.98, P less than 0.005). In the presence of a polyclonal anti-vitronectin receptor antiserum, adhesion of stimulated W256 cells to rIL-1 alpha-treated monolayers was inhibited by 39% +/- 2%, and 3H-2-deoxyglucose release was inhibited by 53% +/- 13%. Immunoblot analysis and immunofluorescence flow cytometry demonstrated that the endothelial cells but not the W256 cells expressed vitronectin receptor (VnR) on their cell surface. The surface expression of VnR by endothelial cells was increased 1.9 +/- 0.1-fold after 4 hours' incubation with rIL-1 alpha. The authors conclude that W256 cell-mediated endothelial damage is dependent on cell adhesion, which, in turn, is partly regulated by the expression of VnR on the endothelial cell surface.

Animals↗