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Previous studies have shown evidence of “spontaneous” injury associated with increased permeability in focal areas of non-atherosclerotic animal aortas. In this study, a mild intimal injury was induced by brief cannulation of the thoracic aorta of rabbits and pigs to examine whether focal aortic plasma accumulation is due to the injury per se or to other effects. Evans blue, a marker of plasma albumin, was used to visualize plasma accumulation. The animals were killed 10 minutes to 5 hours after cannulation. In all animals, blue-stained, sharply demarcated streaks were found in the thoracic aorta. By microscopy, the streaks showed loss of endothelium and, frequently, platelet thrombi with or without fibrin, leucocytes, or red blood cells. The basal platelets were in contact with exposed basement membrane, or with fibres of elastin or collagen. The inner layers of the aortic wall corresponding to the streaks showed signs of oedema. In contrast, unstained areas of the aorta had an intact endothelium, and neither thrombosis nor oedema was present. The observations show that a mild and brief injury of the aortic intima can cause both mural thrombosis and leakage of plasma into the inner layers of the aortic wall. The extent of the leakage appeared to be independent of haemodynamic effects. The injury was similar to, but more severe, than most “spontaneous” lesions.

Objective To determine if there is a relation between aspirin “resistance” and clinical outcomes in patients with cardiovascular disease. Design Systematic review and meta-analysis. Data source Electronic literature search without language restrictions of four databases and hand search of bibliographies for other relevant articles. Review methods Inclusion criteria included a test for platelet responsiveness and clinical outcomes. Aspirin resistance was assessed, using a variety of platelet function assays. Results 20 studies totalling 2930 patients with cardiovascular disease were identified. Most studies used aspirin regimens, ranging from 75-325 mg daily, and six studies included adjunct antiplatelet therapy. Compliance was confirmed directly in 14 studies and by telephone or interviews in three. Information was insufficient to assess compliance in three studies. Overall, 810 patients (28%) were classified as aspirin resistant. A cardiovascular related event occurred in 41% of patients (odds ratio 3.85, 95% confidence interval 3.08 to 4.80), death in 5.7% (5.99, 2.28 to 15.72), and an acute coronary syndrome in 39.4% (4.06, 2.96 to 5.56). Aspirin resistant patients did not benefit from other antiplatelet treatment. Conclusion Patients who are resistant to aspirin are at a greater risk of clinically important cardiovascular morbidity long term than patients who are sensitive to aspirin.

Objective South Asian ethnicity might result in a higher atherosclerotic vascular risk compared with white ethnicity. However, the effect of South Asian ethnicity on clinical outcomes after cardiac surgery is unknown. Thus, we determined whether South Asian ethnicity is a predictor of a poorer outcome after coronary artery bypass grafting. Methods We examined data from 6177 South Asian and white patients who underwent coronary artery bypass grafting at the Toronto General Hospital from January 1994 through June 2003 and used propensity score matching techniques to analyze 917 patients from each group in more detail. Patients were matched for age, sex, body surface area, left ventricular ejection fraction, New York Heart Association class, previous cardiac surgery, number of diseased vessels, and other factors. Independent predictors of operative morbidity and mortality were determined by means of multivariate logistic regression. Results Overall operative mortality was 1.8%. Mortality was higher in South Asian patients than in white patients (2.5% vs 1.1%, P = .02). Postoperative morbidity (eg, myocardial infarction, sepsis, sternal wound infection, postoperative hospital stay, and use of inotropes) also was higher in the South Asian group. In addition to the standard independent predictors of mortality, South Asian ethnicity was an independent predictor of mortality (odds ratio, 3.1; 95% confidence interval, 1.4-6.8). Conclusions These data indicate that South Asian ethnicity per se is an independent predictor of a poorer outcome after coronary artery bypass grafting and suggest that ethnicity is a cardiovascular risk factor that should be considered when assessing clinical outcomes preoperatively before coronary artery bypass grafting or other interventional revascularization procedures.

Postoperative bleeding is one of the most common complications of cardiac surgery.Extensive surgical trauma, prolonged blood contact with the artificial surface of the cardiopulmonary bypass (CPB) circuit, high doses of heparin, and hypothermia are all possible triggers of a coagulopathy leading to excessive bleeding. Platelet activation and dysfunction also occur and are caused mainly by heparin, hypothermia, and inadequate protamine administration. Heparin and protamine administration based on heparin concentrations as opposed to fixed doses may reduce coagulopathy and postoperative blood loss.A better comprehension of the multifactorial mechanisms of activation of coagulation, inflammation, and fibrinolytic pathways during CPB may enable a more effective use of the technical and pharmaceutical options which are currently available.

The endothelium is the monolayer of endothelial cells lining the lumen of all blood vessels. These cells function as a protective biocompatible barrier between all tissues and the circulating blood. Endothelial cells also function as a selective sieve to facilitate bidirectional passage of macromolecules and blood gases to and from tissues and blood. The strategic location of the endothelium allows it to “sense” changes in hemodynamic forces and blood-borne signals and “respond” by releasing a number of autocrine and paracrine substances. A balanced release of these bioactive factors facilitates vascular homeostasis. Endothelial cell dysfunction disrupts this balance, thereby predisposing the vessel wall to vasoconstriction, leukocyte adherence, platelet activation, mitogenesis, pro-oxidation, thrombosis, impaired coagulation, vascular inflammation, and atherosclerosis.1 Our understanding of these endothelial cell responses has led to the development of tests that are believed to reflect endothelial cell dysfunction or integrity in vivo. Given the central role of the endothelium in the development and clinical course of atherosclerosis, endothelial function testing may serve as a useful biomarker of atherosclerosis. Nitric oxide (NO) is the key endothelium-derived relaxing factor that plays a pivotal role in the maintenance of vascular tone and reactivity.2 In addition to being the main determinant of basal vascular smooth muscle tone, NO acts to negate the actions of potent endothelium-derived contracting factors such as angiotensin II and endothelin-1. In addition, NO serves to inhibit platelet and white cell activation and to maintain the vascular smooth muscle in a nonproliferative state. NO is synthesized from l-arginine under the influence of the enzyme NO synthase (NOS). NOS requires a critical cofactor, tetrahydrobiopterin, to facilitate NO production. Tetrahydrobiopterin deficiency leads to an “uncoupling” of NOS that results in the formation of untoward oxidants such as superoxide and hydrogen peroxide (versus NO) with resultant impairment in endothelial function. …

Background: Gender-related differences in morbidity and mortality are well described for coronary artery bypass grafting but are not well understood for combined valve and bypass surgery. Methods We reviewed retrospectively the morbidity and mortality of 1570 consecutive patients who underwent combined valve and bypass procedures at the Toronto General Hospital between January 1990 and October 2000. Results There were 1073 men (68%) and 497 women (32%). The mean ages (± 1 SD) of women and men were 69 ± 9 and 68 ± 9 years, respectively ( P = .02). Of the 1570 total patients, 973 patients (62%) underwent aortic valve and coronary bypass surgery , 481 patients (31%) had mitral valve and coronary bypass operations, and 116 (7%) patients had double or triple valve and coronary bypass operations. Preoperative hypertension ( P = .002), diabetes ( P = .001), and atrial fibrillation ( P = .001) were seen more frequently in women. Body surface area was significantly lower in women ( P = .0001). At presentation, more women were in congestive heart failure (69% vs 58%, P = .001) and in New York Heart Association functional class III or IV (25% vs 19%, P = .001). Although there was no difference in the number of women with three or more diseased vessels (32% vs 38%), only 35% of women received three or more grafts compared with 44% of men ( P = .001). The use of left internal thoracic grafts, although uncommon in the whole study population (36%), was less common in women than in men (26% vs 41%, P = .001). Multivariable logistic analyses for morbidity and mortality showed female gender to be an independent risk factor. Mitral valve replacement, age, left ventricular dysfunction, New York Heart Association classes III and IV, and association of tricuspid valve disease, diabetes, peripheral vascular disease, and preoperative renal failure were found to be independent risk factors for mortality. Conclusion Female gender is an independent risk factor for combined morbidity and mortality during and after combined valve and coronary bypass surgery. As with isolated coronary artery bypass grafting, women undergoing combined procedures have more premorbid conditions, are more often in heart failure, had an equal incidence of triple vessel disease but received fewer grafts than men, and, therefore, were more frequently incompletely revascularized.

SummaryWe investigated the dynamic accumulation of platelets and fibrin after balloon injury of the carotid arteries in rabbits in vivo. In addition, effects of heparin and dipyridamole treatment were also tested. Autologous 99mTc-labelled platelet and 123I-labelled fibrin accumulation was measured at one minute intervals for 4 hours following balloon injury of the carotid artery. Platelet accumulation occurred rapidly, with a ~125% increase occurring within 30 min after injury. There was no further activity for up to 4 hours. This accumulation could be inhibited with an intravenous infusion of PGI2 (500 ng/kg/hr). Fibrin accumulation occurred slowly and continuously over the 4 hour measurement period. Injection of an anti-fibrin antibody inhibited fibrin accumulation. Heparin (25 U/kg/hr for 4 hrs) administration resulted in a significant 82 ± 19% and 68 ± 13% reduction in platelet and fibrin accumulation, respectively. This dose of heparin was associated with a 2-fold prolongation of the aPTT. Dipyridamole (0.45 mg/kg/hr for 4 hrs) resulted in a 46 ± 12% and 70 ± 25% reduction of platelet and fibrin accumulation, respectively. Thus, we demonstrated that the dynamics of platelet and fibrin accumulation following balloon injury in rabbits are very different. The vessel wall continues to be thrombogenic for fibrin up to 4 hours after injury even though platelet accumulation has ceased after one hour. We conclude that the local thrombotic events following balloon injury are complex and that not only platelets but also fibrin is important in regulating responses to injury.

SummaryWe 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.

Thrombin generation and subsequent fibrin deposition occur during cardiopulmonary bypass (CPB) using roller pumps (RPs) despite the administration of high dose heparin. The authors attempted to determine if less thrombin is generated and less fibrin is deposited during CPB using a centrifugal pump (CP). In Part 1 of the experiment, 12 pigs receiving 400 U/kg heparin underwent CPB, including hypothermia, cardioplegia, and aortic cross-clamping, using a CP or RP. Blood samples were collected throughout CPB to measure thrombin generation. At the end of CPB, the amount of fibrin deposited onto each filter was assessed spectrophotometrically. In Part 2, blood samples and arterial in-line filters were obtained from 20 patients undergoing CPB, using either RP or CP, and studied as described previously. The Part 1 results showed that thrombin generation and fibrin deposition in CP pigs were

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)×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 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.

The bleeding time is a readily and easily performed clinical test with immediate results, but there is a degree of subjectivity in its performance and interpretation. We performed a study on 27 volunteers designed to determine the normal range, interobserver reproducibility, and biological variability of the test. Bleeding times in these normal subjects ranged from as low as 129 seconds to as high as 803 seconds. The interobserver variability was 106 seconds (2 standard deviations of the mean of the differences of paired results of repeated measurements), and the coefficient of variation was 18%. For bleeding times taken on the same subjects 6 weeks apart, when the same nurse performed the test at both visits, the difference was 150 seconds (2 standard deviations of the mean of the differences of paired samples) and the coefficient of variation was 27%, and they were essentially the same if a different nurse performed the tests at each visit. There is a wide range in the bleeding times among subjects. However, within individuals there is little biological variability, and most of the difference over time is due to interobserver variability. This suggests that changes in bleeding time are clinically useful in predicting platelet responsiveness in individual patients.

Acetylsalicylic acid (ASA) is effective in preventing strokes, heart attacks and vascular-related events associated with cardiovascular disease (CVD). Notwithstanding, many patients suffer recurrent events while on ASA therapy. During the past decade, a number of investigators have suggested that these patients are unresponsive to ASA or are ‘ASA-resistant’. In the past, this view was met with wide skepticism. Although there is mounting evidence that ASA resistance is a real phenomenon, an understanding of its biological basis and how to measure it is still unclear. The complexity of the problem is discussed below in an attempt to stimulate clinicians and CVD researchers to give serious thought to the ASA resistance problem. It is anticipated that a better understanding of ASA resistance will help us to appreciate its relative importance and its implications in the clinical setting.

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.

SummaryPerformance 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 ≈ 600 ml/2h. 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

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.

The clinical use of heparin in combination with tissue plasminogen activator (t-PA) is based upon the rationale that heparin acceleration of thrombin inhibition by antithrombin III (ATHI) should prevent thrombus growth, allowing for more rapid thrombolysis by t-PA. However, studies both in vitro and in vivo suggest that t-PA+heparin is not synergistic, presumably because t-PA and heparin compete for the same fibrin binding sites. Recenty, we found that dermatan sulphate, which accelerates thrombin inhibition by heparin cofactor II (HCH), prevented fibrin accretion and subsequent thrombus growth more effectively than heparin. Thus, we compared the abilities of heparin and dermatan sulphate to enhance t-PA-induced thrombolysis. Thrombolysis was assessed in 125I-fibrin labelled thrombi in the jugular veins of rabbits. There was no thrombolysis during 4h in saline-treated animals. When rabbits were infused with t-PA (30000 IU/kg/h), thrombolysis was 39±4%, p

SummaryWe 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 deendothelialized 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

SummaryBlood 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.

SummaryWe 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.

The 15-omega-lipoxygenase enzyme in endothelial cells metabolizes endogenous linoleic acid (18:2) into 13-hydroxyoctadecadienoic acid (13-HODE) under basal conditions, i.e., in unstimulated endothelial cells. 13-HODE is thought to regulate the non-adhesivity of the endothelium, contributing to vessel wall/blood cell biocompatibility. We performed experiments, therefore, to determine the relationship between basal levels of cAMP, 13-HODE synthesis, and platelet/endothelial cell adhesion. We found that 13-HODE synthesis increased with elevated cAMP levels and that the elevated 13-HODE levels correlated with increased 18:2 turnover in the triacylglycerol pool. In contrast, neither 18:2 nor arachidonic acid (20:4) turnover in the phospholipid nor prostacyclin (PGI2) production were changed with elevated cAMP levels. Platelet/endothelial cell adhesion was inversely proportional to 13-HODE synthesis. We conclude that intracellular 13-HODE influences platelet/vessel wall interactions, is synthesized from 18:2 released from the endogenous triacylglycerol pool, and that this pathway is modulated by intracellular cAMP levels.

We performed studies to determine the relationship between injured vessel wall thrombogenecity, vessel wall 13-hydroxyoctadecadienoic acid (13-HODE) synthesis and cAMP levels in rabbit treated with salicylate or dipyridamole. Injured vessel wall thrombogenecity was measured as the number of 3 H-adenine labelled platelets adhered to the subendothelial basement membrane exposed by air injury in carotid arteries of rabbits treated orally with salicylate or dipyridamole. Vessel wall 13-HODE was measured by HPLC and vessel wall cAMP was measured by RIA. Vessel wall thrombogenecity was increased two-fold in rabbits treated with salicylate and decreased by half in rabbits treated with dipyridamole. The levels of vessel wall cAMP levels were correlated both with the plasma dipyridamole levels and increases in 13-HODE synthesis. cAMP levels were unaffected by salicylate treatment, but 13-HODE synthesis was decreased. We conclude that there is a significant relationship between vessel wall cAMP levels and 13-HODE synthesis, which in turn, influences subsequent vessel wall thrombogenecity.

SummaryIt has been suggested that protamine sulfate is a poor antidote for the bleeding side-effeets of low molecular weight heparins (LMWHs) in vivo, since protamine sulfate does not completely neutralize the anti-factor Xa activity of LMWHs in vitro or ex vivo. Therefore, we performed experiments to compare directly the abilities of protamine sulfate to neutralize the anticoagulant activities of the LMWH, enoxaparine, and unfractionated heparin ex vivo, with its ability to neutralize the bleeding side-effeets of both compounds in vivo. Bleeding was measured as the amount of blood lost from 5 cuts made in rabbits ears before and after treatment with enoxaparine or unfractionated heparin ± protamine sulfate. Plasma anti-factor Xa and anti-thrombin activities ex vivo, were measured chromogenically. Doses of 400 and 1,500 anti-factor Xa U/kg of heparin and enoxaparine, respectively, were required to enhance blood loss to the same extent. Protamine sulfate completely neutralized blood loss induced by both compounds, but did not neutralize the anti-factor Xa nor antithrombin activities ex vivo. We conclude that protamine sulfate is an effective antidote for the bleeding side-effeets of enoxaparine and unfractionated heparin, despite its inability to completely neutralize their anticoagulant activities.

The majority of studies on fatty acid metabolism, not only via the lipoxygenase pathway, but also via the cyclo-oxygenase pathway, have focused on arachidonic acid. In addition, the majority of the studies have utilized the inhibitor/add-back approach, using exogenous metabolites such as the prostanoids, monohydroxy fatty acids and leukotrienes, and stimulated cells. However, we have explored the possibility that the endothelial cell lipoxygenase metabolite of linoleic acid, specifically, 13 HODE, influences adhesion of a variety of cells implicated in thrombosis, metastasis and inflammation. Albeit, we have until now only demonstrated a correlation between 13 HODE synthesis and maintaining biocompatability between vascular endothelial cells and platelets, tumor cells and leukocytes, our studies provide ample evidence that there is a common relationship between 13 HODE synthesis in vascular wall cells (both endothelial cells and smooth muscle cells), a variety of tumor cells (both human and animal) and leukocytes, and their adhesive interactions with one another. Given that the expression of adhesive sites require an alteration in the cell membrane surface, and, that intracellular 13 HODE inhibition is, in every case, associated with enhanced adhesion, we postulate the intracellular 13 HODE regulates the expression of cell surface adhesive receptors. It is also of interest to note that recent studies in the atherosclerotic-prone Watanabe rabbit are consistant with this hypothesis. Thus, Lawrence et al. [26] found that the atherosclerotic plaque in the Watanabe rabbit wasless, not more, adhesive for platelets than the healthy vessel wall, and Simon et al. [27] found that the same atherosclerotic plaques had increased 15-lipoxygenase activity, consequently, an increased capacity to synthesize linoleic acid into 13 HODE. Studies presently ongoing in our laboratories are focused at identifying the mechanism (s) by which 13 HODE inhibits RGD adhesive site expression and maintains cells less reactive.

Recent studies indicate that chemotherapy is a cause for thrombosis in breast cancer patients. We performed experiments to determine whether the enhanced thrombosis was due, in part, to an effect of chemotherapy on endothelial cell reactivity. Heparinized blood samples were obtained from stage II breast cancer patients receiving monthly adjuvant chemotherapy consisting of cyclophosphamide, epirubicin and 5-fluorouracil. Cultured human endothelial cells were incubated with the plasmas for 2 h, and then the reactivity of the endothelial cells to normal donor platelets was determined isotopically. Endothelial cell reactivity was increased when the endothelial cells were incubated with the post-chemotherapy plasmas. The plasma effect persisted after the chemotherapy drugs were cleared from the circulation, but this plasma effect was abolished when the plasmas were heat-inactivated. Furthermore, the increase in endothelial cell reactivity correlated with the level of interleukin-1 present in the post-chemotherapy plasmas. Finally, the increased endothelial cell reactivity was inhibited by the GRGDS peptide, or by an antibody to the endothelial cell vitronectin receptor. These observations suggest that chemotherapeutic drugs alter endothelial cell reactivity to platelets by inducing the release of interleukin-1 which, in turn, facilitates adhesion molecule expression on the endothelial cell surface. If so, these observations provide a possible explanation for one mechanism which may contribute to the thrombogenic effect seen in breast cancer patients undergoing chemotherapy.

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.

Not all patients with cardiovascular disease (CVD) benefit from long-term aspirin therapy. More importantly, recent evidence indicates that patients who are identified as “aspirin resistant” are at a more than fourfold increased risk of having an adverse cardiovascular event.1–3 These increased risks are seen in patients with different cardiovascular problems who have been classified as aspirin resistant, using a variety of measurements of aspirin resistance. While there are many purported explanations for this—for example, non-compliance, too low an aspirin dose, etc,4 5 it is difficult to refute the evidence that there are numerous platelet function assays, all of which indicate that not all patients’ platelets are dysfunctional after aspirin ingestion—that is, they are aspirin resistant.1–3 6–10 Moreover, the increased risks in these patients are higher than in those patients in whom aspirin yields the expected response, regardless of which test is used.1–3 These increased risks are seen in a wide variety of patient populations with cardiovascular or cerebrovascular disease—that is, in patients with stable CVD, CAD, after percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG), in patients undergoing other vascular procedures and in patients after a stroke.2 However, there is still …

A common factor in the progression of metastasis, atherosclerosis and inflammation is excessive cell growth prompted by both endogenous or exogenous stimuli; e.g. ocogenes, cytokines and thrombin. Excessive cell growth in all of these pathologic responses requires multiple cell cell interactions. There is an abundance of literature suggesting that arachidonic acid metabolites derived from both the cyclooxygenase and lipoxygenase pathways, influence these processes, but it is only recently that our attention has included the importance of linoleic acid metabolism and the relative roles of linoleic and arachidonic acid metabolites in these disease processes.

The Canadian Journal of Cardiology editors have facilitated our understanding of current cardiovascular issues with a varied collection of studies, including solid, evidence-based and up-to-date consensus articles by individuals in collaboration with the Heart and Stroke Foundation of Canada. But equally as important, the editors also provoke our thoughts with articles that may not appear as ‘rigorous’ as we are taught to expect, but ones conveying messages that could significantly impact clinical practice and, therefore, should merit serious consideration. The study by Hobikoglu et al in the current issue of the Journal (pages 201–206) is one of these. Hobikoglu et al followed 140 patients with acute coronary syndrome, first by determining their acetylsalicylic acid resistance status – ie, their platelets responded to acetylsalicylic acid as expected (acetylsalicylic acid-sensitive) or the platelets were not inhibited by acetylsalicylic acid as expected (acetylsalicylic acid-resistant) – and then documenting the risk of adverse clinical events. The study was small – only 140 patients. They did not measure a biomarker of compliance, such as serum salicylate or thromboxane A2. They assessed acetylsalicylic acid resistance using only one assay, namely, the platelet function analyzer PFA-100 (Dade Behring, USA). However, Hobikoglu et al found that acetylsalicylic acid-resistant patients had more than a threefold increased risk of suffering a clinically important event than did acetylsalicylic acid-sensitive patients (OR 3.03; 95% CI 1.06 to 8.62). In 2003, Gum et al (1) reported that among 326 patients with stable cardiovascular disease, acetylsalicylic acid-resistant patients had a 2.9-fold increased risk of suffering a clinically important event (OR 2.86; 95% CI 0.88 to 9.33). Poston et al (2) reported that among 225 patients after coronary artery bypass grafting, acetylsalicylic acid-resistant patients had more than a 2.5-fold increased risk of suffering a clinically important event (OR 2.54; 95% CI 0.89 to 5.17). Chen et al (3) reported that among 117 patients after percutaneous coronary intervention, acetylsalicylic acid-resistant patients had more than a threefold increased risk of suffering a clinically important event (OR 3.82; 95% CI 1.42 to 10.29). Zhang et al (4) reported that among 256 patients after percutaneous coronary intervention, acetylsalicylic acid-resistant patients had more than a 2.8-fold increased risk of suffering a clinically important event (OR 2.87; 95% CI 1.62 to 5.10). Andersen et al (5) reported that among 71 patients after myocardial infarction, acetylsalicylic acid-resistant patients had a 1.8-fold increased risk of suffering a clinically important event (OR 1.79; 95% CI 0.62 to 5.17). Others (6–8) have reported that acetylsalicylic acid-resistant patients have more than a fivefold increased risk of suffering an adverse cardiovascular-related event. All these studies were small. There is no consensus about which acetylsalicylic acid-resistant assay is the most reliable; consequently, the assay varies from one study to the next. The acetylsalicylic acid dose also varies from one study to the next. But wait – despite the obvious limitations of these studies, the message of each is consistently clear and always the same. Regardless of the number of patients studied, the assay used, the dose of acetylsalicylic acid ingested, the clinical entity investigated and the country of study origin, acetylsalicylic acid resistance was found to be associated with a significant increase in risk of adverse events. Specifically, if real, the impact of acetylsalicylic acid resistance on clinical outcome is global and adversely affects all cardiovascular-related disease entities. Moreover, the concept of acetylsalicylic acid resistance has been with us for the past two decades; in other words, the concept of acetylsalicylic acid resistance has stood the test of time. A quick review of the data in the study by Hobikoglu et al and the other studies cited above also provides additional insight: there is a significant dose-response effect. The lower the dose of acetylsalicylic acid, the higher the incidence of acetylsalicylic acid resistance (Table 1). TABLE 1 Dose-related incidence of acetylsalicylic acid (ASA) resistance These results are consistent with those of a previous study that reported on an acetylsalicylic acid dose effect, which, incidentally, was shown to be independent of the inhibition of thromboxane A2 (9). Specifically, acetylsalicylic acid doses of 75 mg/day or more maximally inhibit cyclo-oxygenase activity, thereby blocking thromboxane A2 synthesis, but acetylsalicylic acid resistance can still be detected. The results of a more recent study (10) of 700 patients were consistent with this premise. Thus, the study by Hobikoglu et al remind us once again that we must seriously consider the impact of acetylsalicylic acid resistance on clinical outcome. We may not agree with the assay used, but should we not take steps to ascertain which assay may be better? It is easy for us, as reviewers, to reject studies such as the one by Hobikoglu et al as too small and fraught with limitations. But thanks to the editors at the Journal for allowing the work of Hobikoglu et al to provoke us once again on this important and timely issue. Literally millions of patients throughout the world chronically ingest acetylsalicylic acid to decrease the risk of future untoward cardiovascular events. Clearly, many, and perhaps most, benefit significantly from the acetylsalicylic acid therapy. However, it is difficult to deny the existence of acetylsalicylic acid resistance and its adverse clinical outcome when studies such as the one by Hobikoglu et al repeatedly come to our attention. Is now not the time for industry or academia, as well the Heart and Stroke Foundation of Canada or the Canadian Institutes of Health Research, to facilitate the funding of larger and more rigorous evidence-based studies on acetylsalicylic acid resistance to determine the best way to assess acetylsalicylic acid resistance and its impact on clinical outcome in patients with assorted cardiovascular disease entities? Or can we be assured that acetylsalicylic acid resistance is simply ‘an interesting academic phenomenon with no clinical relevance’, which I have been told on occasion? Tell that to the 16% to more than 30% of patients who do not benefit from acetylsalicylic acid therapy. If acetylsalicylic acid resistance is real, those patients number in the millions per year. Now is the time for us to find out for sure.

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.

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.

Atopy is primarily a condition of hypersensitivity. Atopic disease include atopic eczema, allergic bronchial asthma or hay fever. Atopic dermatitis signs and symptoms are dry itching skin, and eczematous inflammation. Pathophysiology involve: genetics, trigger factors as aeroallergens, superantigens or stress, Th2 lymphocyte cytokine profile (Interleukin (IL)-4,-5,-10) dominance, hyper IgE, monocyte phosphodiesterase increased activity, impaired epidermal barrier function and abnormal linoleic acid metabolism.

Platelet/vessel wall interactions following injury play an important role not only in hemostasis, but also in acute thrombosis and subsequent atherogenesis1. In cardiac surgery, particularly coronary artery bypass grafting (CABG), these platelet/vessel wall interactions are thought to contribute to acute thrombus formation and chronic vessel wall hyperplasia and subsequent vessel wall restenosis. To date, the most common approach to attenuate these responses is the short-term administration of high dose heparin during cardiac surgery (to inhibit thrombin activity) and the long-term administration of low dose aspirin to inhibit platelet thromboxane A2 (TxA2) generation (which is thought to promote platelet aggregation and vasoconstriction 2 . Despite these approaches, acute thrombosis and chronic restenosis remain significant problems in 5 – 40% of these patients within 6–12 months post surgery.

In 1985, we demonstrated that 13-hydroxyoctadecadienoic acid (13-HODE) is synthesized from endogenous linoleic acid triglyceride stores by vascular wall cells via the lipoxygenase pathway1–2. Specifically, we demonstrated that endothelial cells synthesize more 13-HODE than smooth muscle cells, which in turn, synthesize more 13-HODE than fibroblasts. Since then, we and other investigators have demonstrated that both human and animal vascular wall cells, leukocytes and other white cells, tumour cells and epithelial cells all synthesize 13-HODE2–5. In addition, Lamie et al 6 reported that anucleated platelets contain acylated 13-HODE. The levels of endogenous 13-HODE in all of these cells are highest under basal (unstimulated) conditions, and decrease following cell cell interactions; i. e. at a time when the cells respond to injury, pertubation or stimulation. These observations led us to hypothesize that 13-HODE contributes significantly to blood cell vessel wall biocompatability; i. e. 13-HODE appears to downregulate vessel wall thromboresistance, thereby attenuating thrombogenesis and downregulates extravascular invasion during inflammation and metatasis7.

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.

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.

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 (T×B2) during perfusion, whereas inhibition of cyclooxygenase pathway in PMN had no effect either on platelet deposition or on T×B2 levels. In contrast, blockage of platelet cyclooxygenase, which caused almost total inhibition of T×B2, 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.

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.

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.

Platelet interactions with the blood vessel wall following injury influence both the acute thrombotic event (platelet/vessel wall adhesion) and the chronic pathogenesis of arteriosclerosis (chronic vessel wall thickening and occlusion). Platelets are thought not to interact with the healthy intact endothelium, but only to adhere to vessel wall components exposed following endothelial cell damage and vessel wall injury (1–3). When platelets adhere to the injured vessel wall, they release a number of constituents which facilitate further platelet activation (3), activation of the coagulation cascade (1), and/or are mitogenic, facilitating smooth muscle cell proliferation and hyperplasia (4). Endothelial cells also synthesize and release a number of constituents which counterbalance the platelet responses, thereby enhancing fibrinolysis, inhibiting coagulation and preventing platelet aggregation and adhesion (5–7). Recently, with the development of endothelial cell culture techniques and a number of molecular biologic tools, we have learned that platelets and other blood cells actively interact not only with the injured vessel wall, but also with the intact endothelium itself. In this chapter, we will focus on the latter interaction and specifically on endothelial cell fatty acid metabolism and its possible consequence on the reactivity of adhesion molecule receptors on the surface of the intact endothelium which in turn may influence platelet/vessel wall interactions.

The intravascular transport of tumor cells and their attachment to the vessel wall are important steps in metastasis. However, little is known about the intracellular mechanisms which regulate tumor cell adhesion to the vessel wall, particularly, to intact endothelial cells. A number of studies have suggested that circulating tumor cells preferentially adhere to the exposed underlying extracellular matrix rather than to intact endothelial cells (1,2). However, other studies suggest that tumor cells can also adhere to endothelial cells, and that this adherence is dependant upon specific characteristics of the endothelium. It has also been suggested that the metabolic characteristics of tumor cells themselves influence their own adhesion to the vessel wall (3—4). Finally, a number of recent studies suggest that adhesion receptor molecules are expressed following cell stimulation, facilitating cell/cell adhesion (5—7). In this paper we will focus on the possible intracellular regulation of expression of these adhesion molecules by two lipoxygenase derived fatty acid metabolites synthesized from arachidonic and linoleic acids. In particular, we will review the relationship between 13-hydroxyoctadecadienoic acid (13-HODE) derived from linoleic acid and 15-hydroxyeicosatetraenoic acid (15-HETE) derived from arachidonic acid, and tumor cell endothelial cell adhesion.

Cancer cells frequently disseminate via the bloodstream where the endothelium acts as a barrier between the circulating tumor cells and the extravascular tissue. Free radicals generated by environmental factors or host cells can cause endothelial cell injury and we have postulated that such injury could promote the metastasis of intravascular tumor cells. In mice and rats endothelial damage was induced by intravenous cobra venom factor which activates circulating leukocytes. Endothelial cell damage was demonstrated by morphology and by altered vasopermeability. When 125IUdr-labelled cancer cells were injected intravenously, during periods of maximal endothelial cell injury there was a 3 fold increase in retention of these cells in the lung, 24 hours later. After 14 days there was a 3–20 fold increase in the number of metastatic tumors in CoF-treated animals. In both rats and mice, tumor cell localization was reduced 70–80% by pretreatment of the animals with catalase. In mice, endothelial injury induced by bleomycin (120 mg/kg) or by exposure to 90% oxygen for 2–4 days also significantly increased the metastasis of circulating cancer cells. Studies in vitro have demonstrated that rat Walker 256 cancer cells or host cells, when activated with the chemotactic peptide N-fMLP, generate oxygen-derived free radicals and can damage cultured endothelium. When fMLP-activated W256 cells were incubated with 3H-2-deoxyglucose-labelled endothelial cell monolayers, there was a 27% increase in the specific release of isotope within 90 minutes. Damage correlated with tumor cell-induced chemiluminescence and was inhibited by catalase. We conclude that free radical-mediated damage to the microvasculature facilitates the metastasis of circulating cancer cells.

Von Willebrand factor (vWF), fibronectin (FN) and 13-hydroxy-octadecadienoic acid (13-HODE) are known to influence the regulation of the adhesive properties of vascular surfaces. In the present study vWF, FN and 13-HODE were comparatively localized in endothelial cells (EC) and in the extracellular matrix (ECM) produced by EC. An indirect immunofluorescent technique was applied to coverslips containing human EC cultures previously fixed and permeabilized following different procedures: A. Alcohol/acetone; B. Paraformaldehyde alone and C. Paraformaldehyde followed by Triton X-100. vWF was observed inside EC (A), on the ECM produced by EC (B) or in EC and ECM (C) depending on the fixation procedures used. FN was mainly localized in the ECM despite the fixation procedures employed. FN was only seen in relation to cell bodies after strong permeabilization (A). Under our experimental conditions 13-HODE was never found in ECM. This latter antigen was observed randomly dispersed in those preparations fixed with alcohol/acetone, indicating that it is probably extracted by this fixative. 13-HODE was detected in granular shaped structures in EC after permeabilization with detergent (C). These results suggest that the cellular localization of vWF and FN is compatible with an adhesive role related to the abluminal side of ECs. 13-HODE was readily observed after mild permeabilization. This finding would be morphologically consistent with its contribution to the regulation of the vessel wall thromboresistance.

The effect of hydrocortisone on thrombocytopenic bleeding has been studied in rabbits using a jugular vein bleeding-time technique and a microvascular bleeding-time technique. An inverse relationship was found between the bleeding time and platelet count with both techniques in rabbits made thrombocytopenic by either X-irradiation or injection of heterologous platelet antiserum. Hydrocortisone shortened both bleeding times in thrombocytopenic animals when given in single large doses intravenously (25-100 mg/kg), in daily doses (6 mg/kg) intramuscularly, and shortened the jugular bleeding time when applied to the outside of the jugular vein or instilled intraluminally into the vein. This effect was also noted in normal animals. The effect on thrombocytopenic bleeding was dose related. When given daily, the effect was greater when hydrocortisone was given for 10 d than for 5 d. Both indomethacin and tranylcypromine also reduced the jugular vein bleeding time when instilled intraluminally into the jugular vein, whereas exogenously provided arachidonic acid reversed the effect of hydrocortisone but did not reverse the effect of indomethacin or tranylcypromine. Exogenously provided linoleic acid did not have any effect. Perfusion of the vessel segment with prostacyclin (PGI(2)) reversed the effect of intraluminally administered hydrocortisone, indomethacin, and tranylcypromine. Similarly, hydrocortisone, indomethacin, and tranylcypromine all reduced the rate of loss of fluid from a standard wound in isolated vessels emptied of blood and perfused with saline under constant pressure. PGI(2) reversed the action of these three agents, however, arachidonic acid reversed only the effect of hydrocortisone and did not reverse the effect of indomethacin and tranylcypromine. The generation of PGI(2)-like material and 6-keto-prostaglandinF(1) alpha from jugular vein strips was prevented by prior exposure of the animals or vessel wall to hydrocortisone. These results are compatible with the hypothesis that the vessel wall releases smooth muscle-relaxing prostaglandins when injured and that inhibition of prostaglandin formation by hydrocortisone enhances hemostasis by allowing vasoconstriction to be maintained.

Standard heparin, an effective treatment for antepartum thromboembolic disease, is thought to be safe for the fetus since it does not cross the placenta. Recently, a number of low molecular weight heparins have been prepared which have been shown to produce less bleeding than standard heparin for an equivalent antithrombotic effect in experimental animals. These observations suggest that the low molecular weight heparins may also provide superior antithrombotic therapy in antepartum thromboembolic disease. However, it is not known whether the low molecular weight heparins cross the placenta. To determine this, we examined the pharmacokinetics of 125I-labelled standard heparin and a low molecular weight heparin, and their anticoagulant effects in mother and fetus, using a pregnant sheep model. Catheters were inserted into maternal and fetal femoral arteries at 108-119 d gestation (term: 147 d). 1-3 days later the mothers were given a bolus i.v. injection of 5000 anti-Xa units of 125I-labelled standard heparin or low molecular weight heparin, CY 222. Nine serial blood samples were collected over 4 h from both mother and fetus for measurements of radioactivity, anti-Xa activity (chromogenic) and activated partial thromboplastin times. When therapeutic levels of standard and CY 222 heparins were achieved in the mother, there was no detectable radioactivity or anticoagulant effect in the fetus. We conclude that standard heparin and the low molecular weight CY 222 do not cross the placenta in the pregnant sheep.

Prostacyclin (PGI2) is a powerful inhibitor of platelet aggregation, but its role in the pathogenesis of arterial thrombosis is uncertain. We have studied the thrombogenic effect of inhibiting PGI2 production by aspirin (ASA) in carotid arteries of rabbits given 0, 3, 10, or 100 mg ASA/kg either 1, 3, 6, or 20 h beforehand. Platelet accumulation onto injured carotid arteries was enhanced with ASA in a dose of 10 mg/kg. A higher dose of ASA (100 mg/kg) had no further effect. The enhanced thrombogenic effect of ASA persisted for at least 20 h and was associated with a decrease in vessel wall PGI2 production. There was a strong inverse correlation (r = 0.55, P less than 0.01) between PGI2 production and platelet accumulation. The findings suggest that the margin of safety in obtaining an antithrombotic effect of ASA and producing a potential thrombotic effect in arteries may not be as large as predicted by studies using cultured endothelial cells or experimentally induced thrombosis in veins.

Heparan sulphate and dermatan sulphate have both antithrombotic and anticoagulant properties. These are, however, significantly weaker than those of a comparable amount of standard pig mucosal heparin. Antithrombotic and anticoagulant effects of glycosaminoglycans depend on their ability to catalyse the inhibition of thrombin and/or to inhibit the activation of prothrombin. Since heparan sulphate and dermatan sulphate are less sulphated than unfractionated heparin, we investigated whether the decreased sulphation contributes to the lower antithrombotic and anticoagulant activities compared with standard heparin. To do this, we compared the anticoagulant activities of heparan sulphate and dermatan sulphate with those of their derivatives resulphated in vitro. The ratio of sulphate to carboxylate in these resulphated heparan sulphate and dermatan sulphate derivatives was approximately twice that of the parent compounds and similar to that of standard heparin. Anticoagulant effects were assessed by determining (a) the catalytic effects of each glycosaminoglycan on the inhibition of thrombin added to plasma, and (b) the ability of each glycosaminoglycan to inhibit the activation of 125I-prothrombin in plasma. The least sulphated glycosaminoglycans were least able to catalyse the inhibition of thrombin added to plasma and to inhibit the activation of prothrombin. Furthermore, increasing the degree of sulphation improved the catalytic effects of glycosaminoglycans on the inhibition of thrombin by heparin cofactor II in plasma. The degree of sulphation therefore appears to be an important functional property that contributes significantly to the anticoagulant effects of the two glycosaminoglycans.

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.