Biomedical subjects
Publications and source records attributed to M R Buchanan.
We report the successful outcome of allogeneic stem cell transplant (SCT) in a patient with acute lymphoblastic leukaemia (ALL) and pulmonary zygomycosis diagnosed prior to transplant. The lesion was surgically excised and SCT proceeded with antifungal therapy, granulocyte transfusions and G-CSF support during the period of neutropenia.
We compare the relative activities of surface-bound and fluid-phase thrombin and their inhibition by heparin and Intimatan, a novel heparin cofactor II (HCII) agonist. In vitro, we compared the observed amidolytic activities of fluid-phase and surface-bound thrombin with the expected activities based upon 125I-specific activity. In vivo, we compared the inhibitory effects of heparin and Intimatan on thrombin activity bound to injured vessel walls. In vitro, the correlations between observed and expected activities of fluid-phase and surface-bound thrombin, were: r = 0.9974, p < 0.001; and r = 0.9678, p < 0.001; respectively. In vivo, injured vessel wall surface-bound thrombin activity persisted for > 24 h. This activity was not inhibited by heparin, but was inhibited by Intimatan, p < 0.001. We conclude that surface-bound thrombin is as active as fluid-phase thrombin and remains protected from inhibition by heparin, thereby contributing to vessel wall thrombogenicity following injury. In contrast, surface-bound thrombin is inhibited by Intimatan, thereby effectively decreasing vessel wall thrombogenicity following injury in vivo.
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Surface-bound thrombin, which is resistant to inhibition by heparin/antithrombin III (/AT), plays a key role in vessel wall disease. In contrast, surface-bound thrombin is not resistant to inhibition by heparin cofactor II (HCII) and its acceleration of its inhibitory effect by dermatan sulfate. However, the potential use of dermatan sulfate to prevent thrombus formation in vivo is limited by its low specific activity, which in turn, necessitates excessively high doses when given on a gravimetric basis. Recently, a novel HCII agonist, Intimatan, has been synthesized by site-specific sulphation of highly purified dermatan sulfate comprising primarily of L-iduronic acid-4-O-sulphated N-acetyl-D-galactosamine, yielding a 4, 6-O-disulphate compound on the galactopyranose ring with a lower molecular weight, higher solubility, and specific activity than its parent, dermatan sulfate. In this study, we compared the abilities of Intimatan with its parent compound, dermatan sulfate, and with heparin to affect coagulation and to inhibit surface-bound thrombin both in vitro and in vivo, to determine if Intimatan demonstrates a better potential than either other compound in preventing thrombus formation in vivo. Intimatan prolonged the activated partial thromboplastin time (APTT) more effectively than either dermatan sulfate or heparin at comparable antithrombin concentrations. This activity was attributed to the more selective action of Intimatan against surface-bound thrombin in vitro. Intimatan also inhibited thrombin bound to an injured vessel wall surface in vivo more effectively than heparin, i.e., when measured in injured carotid arteries of rabbits injected with Intimatan or with heparin at the time of injury. We conclude that Intimatan effectively inhibits surface-bound thrombin, thereby exhibiting better anticoagulant and antithrombin properties than heparin and dermatan sulfate.
BACKGROUND: Several studies suggest that acetylsalicylic acid (ASA) is less effective in preventing thrombotic events in ASA nonresponder patients. If so, the thrombotic event rate in ASA nonresponders should be higher than in ASA responders. OBJECTIVE: To conduct a prospective, multicentre observational pilot study to determine the thrombotic event rates in ASA responders and nonresponders. PATIENTS AND METHODS: Patients undergoing nonurgent coronary artery bypass grafting (CABG) who were prescribed 325 mg ASA/day were recruited. Patients were classified as an ASA responder or nonresponder based on the ASA effect (or lack thereof) on their bleeding times. All thrombotic events that occurred in the two years following CABG were recorded. These data were stored in a blinded fashion until the last patient follow-up, and then adjudicated by a validation committee. RESULTS: A total of 289 patients recruited at three sites completed the two-year follow-up. Of these patients, 45.3% were classified as ASA responders and 54.7% were classified as ASA nonresponders. Of ASA responders, 6.9% had thrombotic events compared with 9.5% of the ASA nonresponders, but this difference was not significant (P=0.526). CONCLUSIONS: While ASA responder or nonresponder status did not appear to affect the thrombotic event rate in patients undergoing nonurgent CABG, the possibility that ASA responder or nonresponder status affects the thrombotic event rate in more acutely ill CABG patients cannot be excluded.
Intimal hyperplasia after percutaneous transluminal coronary angioplasty (PTCA) or vascular surgical procedures remains a significant problem despite current antithrombotic therapy. The use of the current antithrombotic drugs, namely heparin + chronic aspirin (ASA) +/- oral anticoagulants, is based upon the assumptions that: i) heparin blocks thrombin generation and/or accelerates thrombin inhibition by antithrombin III (ATIII); ii) aspirin acetylates platelet cyclooxygenase, thereby preventing thromboxane A2 (TxA2) synthesis; and iii) oral anticoagulants reduce the availability of vitamin K-dependent procoagulants, thereby reducing the risk of thrombus formation. Albeit beneficial, this approach has a number of shortcomings and limitations: i) when thrombin binds to an injured vessel wall, it becomes resistant to inhibition by heparin/ATIII; thus, surface-bound thrombin remains active, stimulating further thrombus formation, smooth muscle cell proliferation and subsequent hyperplasia; ii) while TxA2 inhibition reduces platelet reactivity, platelets are able to respond to multiple stimuli generated at the time of, or after, vessel wall injury; and iii) heparin, aspirin and the oral anticoagulants all render the patient hemostatically defective and at risk of bleeding. Recent studies suggest that alternate therapeutic approaches can inhibit thrombogenesis more effectively at the time of injury, thereby not only inhibiting hyperplasia more effectively than the currently used drugs, but also reducing (or eliminating) the need for long-term therapy. For example, we suggest that the heparin cofactor II (HCII) catalysts, dermatan sulfate and Intimatan, inhibit surface-bound thrombin more effectively than heparin/ATIII, thereby inhibiting intimal hyperplasia effectively. Their effects are achieved when the drug is given only at the time of injury; i.e. with no further antithrombotic therapy. Other studies indicate that injured vessel wall thrombogenicity can be reduced by pretreatment with Persantine (dipyridamole) or with certain fatty acid supplements which either increase vessel wall cAMP and/or 13HODE synthesis. These increases are associated with decreased vessel wall thrombogenicity, which, in turn, is associated with decreased intimal hyperplasia. Such results suggest that vessel wall repair is achieved more effectively by targeting antithrombotic drugs directly at the vessel wall thrombogenicity per se rather than indirectly by altering the circulating blood cells and systemic coagulant system.
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Surface-bound thrombin which contributes to vessel wall hyperplasia, is resistant to inhibition by heparin/antithrombin III (/ATIII) but not to inhibition by dermatan sulphate/heparin cofactor II (/HCII). To determine the effects of heparin and dermatan sulphate on vessel wall hyperplasia after a first or second injury, rabbit carotid arteries first were injured, using a standard procedure (first injury). Half of the first-injury rabbits were given heparin, dermatan sulphate, or saline, 5 minutes before and at 30-minute intervals over 2 hours post-injury, and then allowed to recover. Four weeks later, the first-injury treated animals were killed and their injured carotid arteries were processed histologically. The remaining untreated first-injury rabbits were also allowed to recover. At 4 weeks, those rabbits were re-anesthetized and their first-injury arteries (which were occluded >75%), were isolated, and vessel wall lumen patency was re-established by endarterectomy (second injury). During this second injury, the animals were treated with heparin, dermatan sulphate, or saline as described above. Four weeks after this second injury, these rabbits were killed and their second injury arteries were processed histologically. Intimal hyperplasia determined histologically, was expressed as an x-fold increase in vessel wall cross-sectional area (i.e., [(media+intima area) media area]). Vessel wall lumen occlusion was expressed as [1-(lumen area/internal elastic lamina area) x 100; % occlusion]. Vessel wall area in the saline-treated animals, increased 2.6+/-1.2 and 2.4+/-1.0 fold respectively, means+/-SD, n = 12, within 4 weeks of the first and second injuries. These increases were due to intimal hyperplasia and associated with 75+/-19% and 79+/-21% occlusion of the vessel wall lumen, respectively. Heparin had little effect, whereas dermatan sulphate (1) decreased hyperplasia by 45% after the first injury and by 47% after the second injury, p<0.008 and <0.03, respectively, and (2) decreased vessel wall occlusion 47+/-12% and 33+/-5% after the first and second injury, respectively. We conclude that (1) dermatan sulphate/HCII may be a useful inhibitor of vessel wall hyperplasia following vessel wall injury, and (2) this effect can be achieved by an acute anticoagulant treatment at the time of injury, unlike heparin/ATIII.
Acetylsalicylic acid (ASA) is currently recommended as an antithrombotic for patients with essential thrombocythemia (ET) who are at an increased risk of thrombotic events. However, ASA is also associated with an increased risk of bleeding in these patients as compared to the risk of bleeding in other patients treated with ASA. Recent data suggest that while ASA inhibits platelet thromboxane A2 (TxA2) synthesis in all individuals, ASA has little effect or inhibits the lipoxygenase pathway (i.e., 12-hydroxyeicosatetranoic acid or 12-HETE synthesis) in some individuals, and enhances 12-HETE synthesis in others. These differential effects are associated with a pronounced prolongation of the bleeding time vs. no prolongation of the bleeding time, respectively, i.e., in ASA responders and ASA nonresponders, respectively. To determine if the increased risk of ASA-induced bleeding seen in ET patients is associated with an effect on 12-HETE synthesis, we compared the relative effects of ASA on the bleeding time, platelet TxA2 and 12-HETE synthesis, and platelet aggregation and adhesion in ET patients and healthy volunteers. ASA (300 mg, taken orally) prolonged the bleeding time in 82% of the ET patients but only 27% of the healthy volunteers although platelet TxA2 synthesis and ADP- and collagen-induced aggregation were inhibited significantly in both groups. In contrast, platelet 12-HETE synthesis was unchanged and platelet adhesion was decreased in those patients and volunteers whose bleeding times were prolonged by ASA, whereas platelet 12-HETE synthesis was increased significantly and platelet adhesion was unaffected in those patients and volunteers whose bleeding times were not prolonged, and in some cases shortened by ASA. These results confirm previous data that demonstrate that ASA has different effects on platelet 12-HETE synthesis and platelet adhesion in different individuals, i.e., inhibitory or no effect in ASA responders (in whom ASA prolonged bleeding) vs. enhancing effects in ASA nonresponders (in whom ASA did not prolong bleeding). These results also indicate that there is a greater percentage of ASA responders in patients with ET than that seen in the general population, a difference that is associated with an effect of ASA on the lipoxygenase pathway. This may explain the increased bleeding side effects seen in the ET patient population.
In previous studies, we reported that vascular wall cells such as endothelial cells metabolize linoleic acid to 13-hydroxyoctadecadienoic acid (13-HODE) via the 15-lipoxygenase pathway. Endothelial cell 13-HODE levels vary inversely with endothelial cell reactivity to platelets, which, in turn, varies directly with the expression of the vitronectin receptor (VnR) on the apical surface of endothelial cells. We and others have also found that tumour cell adhesivity is dependent, in part, upon the relative amounts of intracellular 13-HODE and the arachidonic acid monohydroxide(s), 12- and/or 15-hydroxyeicosatetraenoic acids (12-, 15-HETE). In addition, we and others have found that platelet adhesivity is dependent upon the intraplatelet level of its major lipoxygenase metabolite, 12-HETE. Finally, we have demonstrated that 13-HODE and VnR co-localize in nonadhesive endothelial cells but dissociate following endothelial cell injury, at which time, the VnR relocates on the endothelial cell apical surface. These data suggest to us that lipoxygenase-derived monohydroxides regulate the ability of various receptors to recognize their specific ligands. The latter data also suggest that these monohydroxides act directly by a physiochemical mechanism. The present study supports this possibility. Thus, we demonstrate that 13-HODE downregulates VnR binding with vitronectin (Vn) > fibronectin (Fn) > fibrinogen (Fgn), whereas 12- and 15-HETE upregulate specific VnR/ligand binding, using purified VnR/liposomes and purified ligands in an adhesion assay; and that 12- and 15-HETE upregulate GPIIb/IIIa:liposome binding of Fgn > Fn > Vn. We conclude that cell-specific monohydroxides influence cell-specific receptor-ligand binding directly through a physiochemical mechanism.
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Depletion of internal Ca2+ stores causes capacitative Ca2+ entry which occurs through non-selective cation channels sensitive to blockade by SK&F 96365. Recently, alkaloids of Chinese herbal medicinal origin, tetrandrine and hernandezine, have been shown to possess actions including inhibition of Ca2+ channels in non-excitable cell types. In this study, we compared the actions of these novel inhibitors to those of SK&F 96365 in fura-2-loaded endothelial cells from human umbilical vein and bovine pulmonary artery. Depletion of Ca2+ from the internal stores was accomplished in Ca(2+)-free medium using an endoplasmic reticulum Ca2+ pump inhibitor, cyclopiazonic acid (CPA) or receptor agonists, histamine and bradykinin. Stimulation with histamine or bradykinin caused a marked and rapid transient increase in Ca2+ signal whereas CPA caused a smaller amplitude increase of longer duration. Restoring Ca2+ to the medium caused marked and sustained increases in the fluorescence indicating movement of Ca2+ into the cytosol presumably stimulated by the emptied Ca2+ stores. SK&F 96365 as well as tetrandrine and hernandezine antagonized depletion-induced Ca2+ entry. The results suggest that these putative inhibitors interact with Ca2+ entry triggered by depletion of the internal Ca2+ stores and their action is presumed to be on the non-selective cation channels. Their effectiveness may be enhanced by the mechanisms which lead to the opening of the Ca2+ influx channel.
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OBJECTIVE: To determine whether acetylsalicylic acid (ASA) inhibits hemostasis and platelet function in some individuals (ASA responders) but not in others (ASA nonresponders). DESIGN: In this two-part study, part 1 was a randomized, double-blind crossover study of the effects of various single doses of ASA (80 to 1300 mg) on primary hemostasis and platelet function. Part 2 was a prospective cohort study of the effects of a chronic dose of ASA (325 mg) on primary hemostasis and platelet function. SETTING: A hospital research laboratory and a cardiac care ward. SUBJECTS: Part 1: 10 healthy volunteers (five male, five female). Part 2: 40 consecutive patients undergoing elective coronary artery bypass grafting (CABG). RESULTS: Part 1: ASA, in a dose-related manner, prolonged the bleeding time in 60% of volunteers (ASA responders), which was associated with decreases in platelet thromboxane (Tx) A2 and 12-hydroxyeicosatetraenoic acid (12-HETE) synthesis and in platelet aggregation and adhesion. However, in volunteers whose bleeding time was not prolonged (ASA nonresponders), platelet 12-HETE synthesis and platelet adhesion were unchanged or increased (P < 0.001), despite platelet TxA2 and platelet aggregation being inhibited. Part 2: similarly, 58% of the CABG patients were ASA responders and all of their platelet biochemistry and function tests were inhibited, while in the CABG patient ASA nonresponders (no prolongation of bleeding time), platelet 12-HETE and platelet adhesion were increased (P < 0.001).
We determined the extent and duration of activation of coagulation during the first 24 hours after carotid endarterectomy. Serial blood samples were collected before, during and after surgery from 10 patients with severe stenosis (> 70%) undergoing carotid endarterectomy with heparinization. Platelet poor plasmas prepared from these samples, were analysed for activated prothrombin fragment F1 + 2 and thrombin-antithrombin III (TAT) levels, using commercially available ELISA kits. F1 + 2 and TATs were measured as indices of thrombin generation and inhibition respectively. Baseline F1 + 2 and TAT levels were 1.19 +/- 0.27 nMol/ml and 17 +/- 10 pMol/ml, respectively. Neither the F1 + 2 nor TAT level increased during surgery at a time when the patients were heparinized. However, both the F1 + 2 and TAT levels increased significantly within 3 hours after surgery and after the heparin had been neutralized with protamine, (p < 0.01). Moreover, both the F1 + 2 and TAT levels remained elevated in 75% of the patients for at least 24 hours, p < 0.01. We conclude that i) thrombin generation is significant post surgery in patients undergoing carotid endarterectomy despite their receiving heparin during surgery; ii) heparin may not be the ideal anticoagulant for carotid endarterectomy; and iii) persistent thrombin generation may contribute to early post-endarterectomy ischemic events.