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Biomedical subjects

M R Buchanan

Publications and source records attributed to M R Buchanan.

At least 91 records · Page 5Linked to original sources
PubMed1987

Effect of nafazatrom on platelet function and release: relationship to symptomatic episodes in patients with peripheral vascular disease.

We studied the effect of nafazatrom on plasma prostacyclin (PGI2) levels, platelet function, and thromboxane B2 (TxB2), and 12-hydroxy-eicosatetraenoic acid (12-HETE) production and clinical improvement in 12 patients with peripheral vascular disease (PVD) by means of a double-blind crossover trial of placebo, 800 or 1600 mg of nafazatrom four times daily for 1 week, with intervening 2-week washout periods. Plasma PGI2 levels were measured as 6-keto-PGF1 alpha by radioimmunoassay. Platelet function ex vivo was measured as collagen and adenosine diphosphate (ADP)-induced platelet aggregation, release of 12-HETE and thromboxane A2 (measured as TxB2), and was determined by high-pressure liquid chromatography (HPLC) and radioimmunoassay, respectively. The plasma 6-keto-PGF1 alpha levels were unaffected by nafazatrom treatment (p greater than 0.25). Nafazatrom treatment had no effect on TxB2 production, but significantly altered the production of the platelet 12-HETE (p less than 0.05). There was a significant association between the changes in 12-HETE production and clinical improvement. These results suggest that the mechanism of action of nafazatrom is in part related to the inhibition of platelet function via the lipoxygenase pathway, independent of PGI2 stimulation.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1987

Primary thrombocythaemia in childhood: symptomatic episodes and their relationship to thromboxane A2, 6-keto-PGE1 and 12-hydroxy-eicosatetraenoic acid production: a case report.

In a child with primary thrombocythaemia, observations have been made over a period of five years, during which, transient apparently thrombotic events occurred in the central nervous system on several occasions. Spontaneous platelet aggregation was noted and deaggregation took place even after exposure to ADP in vitro. Associated findings included pronounced elevation in plasma levels of 6-keto-PGF1 alpha and 6-keto-PGE1, the latter described for the first time. Production of 12-HETE by platelets was markedly reduced, probably reflecting lipoxygenase deficiency which has been reported in other myeloproliferative disorders. It has been suggested that 12-HETE is a natural inhibitor of thromboxane synthetase, so the further finding of enormous generation of TxA2, measured as TxB2, by this patient's platelets may be explicable. It is suggested that the increase in TxA2 is responsible for spontaneous platelet aggregation. In response to these massive events, there is a production of 6-keto-PGE1 which in turn, promotes platelet deaggregation. Administration of aspirin resulted in symptomatic relief and complete inhibition of TxB2 production.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1986

Heparin fractions with high and low affinities to antithrombin III are cleared at different rates.

The clearance characteristics of standard heparin (SH) and its fractions with high and low affinity to antithrombin III (HAH and LAH respectively) were studied by injecting 125I-labelled SH, HAH and LAH intravenously into the rabbits in increasing doses. Serial blood samples were then collected from an indwelling cannula for measurement of clearance, based both on radioactivity and anticoagulant activity. For equivalent weights injected, the radioactivity of HAH was cleared more slowly from the circulation than that of either SH or LAH. The radioactivity of SH, in turn, was cleared more slowly than that of LAH. The clearance of HAH, measured both by radioactivity and by anticoagulant activity were similar, whereas the clearance of the radioactivity of SH was more rapid than its anticoagulant activity (i.e. anti-factor Xa activity). These observations suggest that the radioactivity clearance curves of SH reflect a "net" estimation of the more complex clearance curves of the different heparin moieties whereas the anticoagulant clearance curves of SH reflects the clearance of the anticoagulant activity of a specific heparin fraction, in this case, the clearance of the anti-factor Xa activity of HAH. These differences suggest that the HAH:LAH ratio is, at any given time, greater in vivo than the same ratio measured in vitro.

Animals↗
PubMed1986

A low molecular weight heparin alters the fetal coagulation system in the pregnant sheep.

Standard heparin and a LMWH, CY222 do not cross the placenta nor alter fetal coagulation when injected into the pregnant ewe. We found that another LMWH, Pharmuka-10169 (PK-10169) alters fetal coagulation without crossing the placenta in the pregnant sheep. To characterize this anticoagulant we measured the in vitro and in vivo effects of 125I-PK-10169 in maternal and fetal plasmas following administration of PK-10169 to the mother or fetus. The fetal anticoagulant activity was not neutralizable by protamine sulphate and was attributable to the inhibition of thrombin but not factor Xa. In vitro, the fetal anticoagulant activity had properties similar to dermatan sulphate; both catalyzed the inhibition of thrombin but not factor Xa by sheep plasma; and neither was neutralizable by protamine sulphate. These effects were due to the enhanced neutralization of thrombin by heparin cofactor II. We conclude that PK-10169 does not cross the placenta, but does induce the release of an endogenous dermatan sulphate-like substance which alters fetal coagulation.

Animals↗
PubMed1986

Role of lipoxygenase metabolism in platelet function: effect of aspirin and salicylate.

Aspirin inhibits thromboxane A2 (TxA2) production whereas its salicylate moiety inhibits 12-hydroxy-eicosatetraenoic acid (12-HETE) production in the platelet. The significance of the latter effect on platelet function is unclear. We examined the effects of aspirin and salicylate on (i) platelet/collagen adhesion using 3H-adenine-labelled human platelets and collagen-coated discs, (ii) platelet aggregation induced by thrombin, collagen, ADP and arachidonic acid, and (iii) platelet TxA2 and 12-HETE synthesis as measured by radioimmunoassay and high pressure liquid chromatography respectively. Aspirin (50 uM) decreased platelet aggregation and increased platelet adhesion. The decrease in aggregation was associated with inhibition of TxA2 production and the increase in adhesion was associated with enhanced 12-HETE production. Salicylate had the opposite effects. Platelet aggregation was increased and platelet adhesion decreased. The increased aggregation was associated with enhanced TxA2 production and the decrease in aggregation was associated with inhibition of 12-HETE production. These observations suggest that 12-HETE facilitates platelet adhesion which can be altered by salicylate treatment.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1986

Respective role of antithrombin III and heparin cofactor II in the in vitro anticoagulant effect of heparin and of various sulphated polysaccharides.

The in vitro anticoagulant effects of standard heparin (SH) and of seven other sulphated polysaccharides (SPS) were investigated by measuring activated partial thromboplastin time (APTT) prolongation of normal plasma and of plasmas selectively depleted of antithrombin III (AT III), of heparin cofactor II (HC II) and of both heparin cofactors. This allowed the determination of the relative contribution of each of the two heparin cofactors to the SPS anticoagulant effect. The SPS varied in their relative activities as catalysts of thrombin inhibition by purified AT III or HC II. The anticoagulant activities of heparin and dermatan sulphate were primarily attributable to their ability to enhance thrombin inhibition by AT III and HC II respectively. Heparin had an additional minor anticoagulant activity which was independent of both AT III and HC II. Pentosan polysulphate, high molecular weight dextran sulphate, heparin with low affinity for AT III and a sulphated heparin derivative had weaker anticoagulant activities in normal plasma than standard heparin. The anticoagulant activities of these last four SPS in plasma depleted of both AT III and HC II were similar to their respective activities in normal plasma. This suggests that these SPS act by directly preventing thrombin generation rather than by enhancing thrombin inhibition.

Antithrombin III↗
PubMed1986

The haemorrhagic and antithrombotic effects of dermatan sulphate.

Heparin and dermatan sulphate are effective antithrombotic agents but the clinical use of heparin is complicated by haemorrhage. The haemorrhagic effect of dermatan sulphate is unknown. In this study we compared the antithrombotic, haemorrhagic and anticoagulant effects of heparin and dermatan sulphate in rabbits. The antithrombotic effect was measured as prevention of venous thrombus formation. The haemorrhagic effect was measured as 51Cr-blood loss from standardized cuts in rabbit ears. The anticoagulant effect was measured as changes in the APTT, TCT and circulating anti-factor Xa level, and the formation of 125I-thrombin/inhibitor complexes ex vivo. The effect of heparin and dermatan sulphate on collagen-induced platelet aggregation was measured ex vivo. Maximal antithrombotic effects of heparin and dermatan sulphate were achieved with 70 and 500 micrograms/kg respectively. A 20-fold increase in heparin dose caused an 8-fold increase in blood loss and higher doses (40- and 80-fold increases) caused further dose-related increases in blood loss (13- and 35-fold increases respectively). In contrast, a 20- to 40-fold increase in the antithrombotic dose of dermatan sulphate did not increase blood loss and an 80-fold dose increase caused only a 7-fold increase in blood loss. There was no relationship between the antithrombotic and haemorrhagic effects of either heparin or dermatan sulphate and their anticoagulant activities. In contrast, there was a relationship between the dose-related enhancement of blood loss by these glycosaminoglycans and the inhibition of collagen-induced platelet aggregation ex vivo. These results suggest that dermatan sulphate is less haemorrhagic than heparin at equivalent antithrombotic doses, and that the haemorrhagic effect is associated with a glycosaminoglycan-induced platelet defect.

Animals↗
PubMed1986

Invasive melanoma in the donor and recipient sites of a Hutchinson's freckle transplanted in a split skin graft.

An extraordinary case of synchronous development of invasive melanoma in the donor and recipient sites of a split skin graft is reported. The histological features suggest transplantation of a Hutchinson's melanotic freckle (lentigo maligna) at operation 10 years previously as the probable cause. The case is particularly interesting in that the invasive melanoma at both sites was at a similar stage despite a considerable difference in exposure to sunlight in the long postoperative period, perhaps discounting this factor as a precipitating cause of melanoma in Hutchinson's melanotic freckle (lentigo maligna melanoma).

Humans↗
PubMed1986

Mechanisms for inhibition of the generation of thrombin activity by sulfated polysaccharides.

Three mechanisms by which sulfated polysaccharides act as anticoagulants and possibly as antithrombotic agents have been described. These are the two heparin cofactor-dependent mechanisms involving the catalysis of the inhibition of various proteases of coagulation by either antithrombin III or heparin cofactor II. The third is a heparin cofactor-independent mechanism involving the inhibition of formation of prothrombinase and tenase complexes. Four sulfated polysaccharides previously shown to have anticoagulant and antithrombotic effects were assessed to determine which of the three mechanisms operate in the expression of their anticoagulant effects. To do this, [125I]prothrombin was added to undiluted human plasma, and the inhibition of [125I]prothrombin activation, or the catalysis of the formation of thrombin-inhibitor complexes was determined in plasma containing one of the four sulfated polysaccharides. Prothrombin activation was demonstrated by the formation of [125I]prothrombin fragment 1.2 and [125I]thrombin. The effect of the thrombin-specific inhibitor, D-Phe-L-Pro-L-ArgCH2Cl (PPACK), on prothrombin activation was also investigated to determine the role of thrombin-dependent feedback reactions on efficient prothrombin activation. Use of PPACK with sulfated polysaccharides also facilitated estimation of the role of the heparin cofactor-independent effects of sulfated polysaccharides on prothrombin activation. Three concentrations of each of the sulfated polysaccharides were used: 0.66, 6.6, and 66 micrograms/ml of plasma. PPACK (1.0 X 10(-6)M) completely inhibited both intrinsic and extrinsic prothrombin activation. The inhibition of prothrombin activation caused by PPACK was abolished when thrombin was added to the plasma before PPACK. These observations indicate that the presence of trace thrombin activity is critical for efficient prothrombin activation by both the intrinsic and extrinsic pathways. All three concentrations of standard heparin completely inhibited the intrinsic activation of prothrombin. This inhibition was only partially abolished when thrombin was added to the plasma before heparin, indicating that heparin inhibits prothrombin activation both by catalyzing the inhibition of thrombin activity and by a heparin cofactor-independent mechanism. Heparan sulfate did not inhibit intrinsic prothrombin activation but catalyzed the inhibition of the thrombin generated by the formation of thrombin-antithrombin III complex. Dematan sulfate inhibited intrinsic prothrombin activation only at the highest concentration. At the two lower concentrations, dermatan sulfate catalyzed formation of thrombin-heparin cofactor II.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Chloromethyl Ketones↗
PubMed1985

13-Hydroxyoctadecadienoic acid is the vessel wall chemorepellant factor, LOX.

We have previously reported that endothelial cells synthesize a cytosol-associated, lipoxygenase-derived metabolite, LOX, which acts as a chemorepellant and, in so doing, maintains the vessel wall thromboresistance. In this study we demonstrate that LOX is a 13-hydroxylinoleic acid (13-OH-18:2) derived from linoleic acid and identical to 13-hydroxy-9-cis,11-trans-octadecadienoic acid, as measured by both reverse phase high pressure liquid chromatography and gas chromatography/mass spectrometry. In addition, we demonstrate that 13-OH-18:2 is produced in significantly greater quantities by endothelial cells than by smooth muscle cels or by fibroblasts. Furthermore, we demonstrate that 13-OH-18:2 is produced in microgram amounts under basal conditions and is decreased by thrombin, calcium ionophore, and trypsin stimulation. And finally, we demonstrate that endothelial cells do not synthesize any significant amounts of lipoxygenase-derived arachidonic acid metabolites either under basal or stimulated conditions unless exogenous arachidonic acid is added. These observations indicate that the major lipoxygenase-derived, chemorepellant metabolite produced by the endothelial is 13-hydroxy-9-cis,11-trans-octadecadienoic acid.

Antithrombins↗
PubMed1985

The thrombolytic and hemorrhagic effects of tissue type plasminogen activator: influence of dosage regimens in rabbits.

Tissue type plasminogen activator (t-PA) is an effective thrombolytic agent in experimental animals and in humans. We have previously observed that the thrombolytic effect of t-PA persists beyond its time of clearance from the circulation and that wound bleeding induced by a high dose infusion of t-PA is delayed for at least one and a half hours after commencing the infusion. These observations suggest that improved thrombolysis may be obtained with t-PA by infusing a high dose over a short period of time. To test this hypothesis we compared the thrombolytic and hemorrhagic effects of t-PA infused over 240, 60, 30 and 15 minutes in the rabbit. A 4 hour infusion of 30,000 U/kg of t-PA produced 36% thrombolysis. The same dose of t-PA infused over 60, 30 and 15 minutes produced 87%, 88% and 96% thrombolysis, respectively (p less than 0.01). The 1 hour infusion of 30,000 U/kg of t-PA produced a significant increase in blood loss relative to saline infusion (p less than 0.01). The same dose infused over 30 and 15 minutes did not produce significantly more bleeding than saline. A higher dose of t-PA, 60,000 U/kg, infused over 4 hours, produced 95% thrombolysis, but was associated with a significant increase of blood loss (p less than 0.001). Our findings suggest that improved thrombolysis with t-PA with minimal bleeding side-effects is achieved in rabbits when t-PA is administered in a relatively high dose given over a short period of time.

Animals↗
PubMed1985

Effects of heparin, its low molecular weight fractions and other glycosaminoglycans on thrombus growth in vivo.

Low molecular weight heparin fractions (LMWH) are less hemorrhagic but are as effective as standard heparin (SH) in preventing experimentally-induced venous thrombosis. The effect of LMWH in preventing extension of established thrombi is unknown. We have compared the effects of two LMWH's (CY, PK), and a low molecular weight heparinoid (a dermatan/heparan/chondroitin mixture, OH) with SH on the prevention of extension of established venous thrombi, by measuring their ability to inhibit the accretion of 125I-fibrin onto venous thrombi pre-formed in rabbit jugular veins. Anticoagulant activity was assayed ex vivo by the APTT and a chromogenic anti-Xa assay, and the antithrombotic effect of these glycosaminoglycans was related to their anticoagulant effects. Autologous thrombi were formed in both jugular veins of each rabbit. The rabbits were then injected with 125I-fibrinogen and treated with a bolus dose of glycosaminoglycan or saline, followed by a continuous infusion for 4 hours. All four glycosaminoglycans significantly inhibited 125I-fibrin accretion (p less than 0.001). SH, CY and PK were equipotent at doses of 42.5-62.5 anti-Xa U/kg/hr in preventing fibrin accretion by 50%. Higher doses had no further effect. OH was significantly more potent than the other three glycosaminoglycans at any given dose (p less than 0.005). There was no correlation between the antithrombotic effect and the anticoagulant effects. We conclude that these LMWH's are as effective as SH in preventing extension of established thrombosis.

Animals↗
PubMed1985

Endothelial cells produce a lipoxygenase derived chemo-repellent which influences platelet/endothelial cell interactions--effect of aspirin and salicylate.

We performed experiments to determine whether endothelial cells synthesize phospholipid metabolites via the lipoxygenase pathway and whether these metabolites influence platelet/vessel wall interactions. Monolayers of cultured human endothelial cells were incubated with 14C-arachidonic acid and their cyclo-oxygenase and lipoxygenase metabolites were extracted and identified by radioimmunoassay, thin layer chromatography and high performance liquid chromatography. We found that in addition to the membrane-associated production of PGI2, endothelial cells synthesized a cytosol-associated metabolite, LOX, which was presumably derived through the lipoxygenase pathway. Inhibition of LOX was associated with an increase in PGI2 production and inhibition of PGI2 with an increase in LOX production. Under either condition, platelet adhesion to cultured endothelial cells was significantly decreased. In contrast, when both PGI2 and LOX production were inhibited, platelet adhesion to endothelial cells was enhanced. Furthermore, when LOX was bound to a thrombogenic surface, platelet adhesion was significantly decreased whereas when arachidonic acid or 12-HETE was bound to the surface, platelet adhesion was increased. We conclude that endothelial cells produce not only a cyclo-oxygenase metabolite, but also a lipoxygenase metabolite, both of which influence platelet/endothelial cell interactions.

Adenine↗
PubMed1985

Scanning electron microscopy of normal rabbit aorta: injury or artifact?

The reported morphology of both normal and "injured" aortic endothelium differs significantly from one study to another. To better understand these disparities, we examined the effects of manipulating the preperfusion conditions on the subsequent morphology of normal aortic endothelium in the rabbit. Glutaraldehyde perfusion without any preperfusion with an electrolyte solution, induced vasoconstriction and the accumulation of cellular and plasma protein on the luminal surface of the aorta. Prolonged preperfusion by Ringer-Locke solution and to a lesser extent, by Krebs-Henzleit solution or modified Eagles medium, induced changes in the endothelium similar to those reported as the response to injury induced by other stimuli. Profound vessel wall alterations also occurred in animals which were shocked (through acute blood loss) or killed (by overdose of anesthetic) prior to fixation. These observations may explain, in part, the discrepancies in previous descriptions of the appearance of "normal" rabbit aortic endothelium in SEM, and suggest that the type and duration of the preperfusion must be considered to avoid morphological artifacts.

Animals↗