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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.

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

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.

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 …

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.

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.

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.

The effect of experimental hematomas on serum levels of fibrinogen- related antigen (FRA) was studied in rabbits. Homologous blood used for production of hematomas was mixed with 131I-fibrinogen, and the measurement of FRA was made using a radioactive technique (representing FRA from hematoma) and immunologically (representing total FRA). Total body radiation of animals bearing hematomas containing 131I-fibrinogen decayed slowly (mean t 1/2 = 189 hr) over the first 2–3 days and then rapidly (mean t 1/2 = 43 hr) during the subsequent days. Serum FRA levels measured immunologically rose transiently from 4 to 11 mug/ml during the slow phase of hematoma resolution, and returned to baseline levels during the rapid phase of resolution. The appearance of a small amount of protein-bound radioactivity in the blood of animals with hematomas paralleled the time course of the changes in immunologically determined FRA, but, even at its peak, circulating levels only averaged 0.36% of the administered tracer. From these observations, it seemed unlikely that the dissolution of fibrin which may form as the result of bleeding into tissues would significatnly contribute to levels of circulating FRA.

There are a number of reports which suggest that the antithrombotic effect of aspirin is limited to males. It is unclear whether this effect is due to sex-related differences in the effect of aspirin on platelets, the vessel wall, or the pharmacokinetics of aspirin. To test these possibilities we examined the sex-related differences in (1) vessel wall PGI 2 release and its inhibition by and recovery from aspirin in rabbits; (2) the effects of aspirin on platelet aggregation, thromboxane B 2 and beta-thromboglobulin (BTG) release in man, and (3) the pharmacokinetic characteristics of aspirin, in both rabbits and man. Vascular wall PGI 2 measured as 6-keto-PGF 1α , was not different in male and females rabbits, and was inhibited to a similar extent by identical concentrations of aspirin. The duration of this inhibitory effect was also the same in males and females. The pattern of inhibition of collagen-induced platelet aggregation, and collagen-induced thromboxane B 2 and BTG release by aspirin were not different in either sex. There was, however, a sex-related difference in a number of pharmacokinetic characteristics of aspirin both in rabbits and man. Thus, aspirin was absorbed more rapidly, distributed in larger apparent volume and was hydrolysed more rapidly in females. These observations suggest that the sex-related differences in the antithrombotic effects of aspirin seen in clinical studies are not due to differences in the effects of aspirin on the inhibition of platelet function mediated by the inhibition of cyclo-oxygenase in either the platelet or the vessel wall. An effect of aspirin on platelet function independent of the inhibition of cyclo-oxygenase has been described and it is possible that this effect may be influenced by sex-related differences in the pharmacokinetics of aspirin.

Thrombolytic agents may be useful in acute pulmonary embolism, but their optimal dosage remains uncertain. We have examined the relative efficacy of heparin and different doses of streptokinase, either alone or in combination, in acute experimental pulmonary embolism. A standardized massive embolus of autologous blood clot incorporating canine [(125)I]-fibrinogen was given to 40 dogs; the degree of resolution after 24 h was quantitated by measuring the radioactivity in the lungs and was compared with detailed postmortem observations. The amount of residual embolus was 49% in control animals, 28% after heparin (200 U/kg loading dose and 800 U/kg/24 h maintenance dose), and 6% after high dose streptokinase (250,000 U loading dose and 100,000 U/h maintenance dose); it was 31% after low dose streptokinase (25,000 U loading dose and 10,000 U/h maintenance dose), 7% after low dose streptokinase with heparin, 14% after very low dose streptokinase (5,000 U/h without a loading dose) with heparin, and 9% after short course streptokinase (250,000 U loading dose and no maintenance dose) with heparin. The combination of heparin and low doses or brief courses of streptokinase appeared to be synergistic and produced as much resolution as did standard high dose streptokinase alone. The enhanced resolution of pulmonary emboli in heparin-treated animals may have been due to the prevention by heparin of further deposition of fibrin on the embolus. It appears that dosage regimens of thrombolytic therapy other than those in current use may be worthy of clinical examination.

Aspirin, in doses which inhibit platelet thromboxane A2 production, prolongs the bleeding time but this effect on the bleeding time is lost when doses of aspirin which also inhibit vessel wall prostaglandin I2 (PGI2) production are used. PGI2 is both a potent inhibitor of platelet aggregation and a powerful vasodilator. We have investigated the contribution of the vascular effect of PGI2 on hemostasis by studying the effect of high concentrations of aspirin on the jugular vein bleeding time in severely thrombocytopenic rabbits and on the loss of non-platelet-containing fluid from standard puncture wounds in aspirin-treated veins perfused under constant pressure. After aspirin treatment, the bleeding time was significantly shortened in both normal and thrombocytopenic rabbits. This effect was associated with a decreased production of PGI2-like material by the vessel wall and a reduction in the volume of fluid lost from the standard puncture wound in the jugular vein. The effect of aspirin on the bleeding time and on PGI2 production was relatively short-lived and the bleeding time returned to normal within 2–3 hours. These observations indicate that PGI2 can influence hemostasis by mechanisms independent of platelet aggregation.

Aspirin inhibits platelet function by acetylating platelet cyclo-oxygenase. When aspirin is administered in doses as low as 40-160 mg per day, it inhibits platelet cyclo-oxygenase activity by more than 80%. The effect of aspirin on platelet function is maintained for the life-span of the platelet and there is evidence that aspirin also acetylates platelets before they are released in the circulation and while they are still within megakaryocytes. Aspirin also inhibits the synthesis of PGI2 by vascular wall cells but compared to the platelet, this vessel wall effect is relatively short-lived and requires slightly larger doses of aspirin. In vivo studies in rabbits indicate that very high doses of aspirin are thrombogenic. However, there is no evidence that aspirin is thrombogenic in man even when administered in high therapeutic doses. The optimal antithrombotic dose of aspirin has not yet been determined. Clinically, impressive results have been obtained with low doses of aspirin (ranging from 100 to 300 mg per day) in preventing aorta coronary bypass thrombosis, in patients undergoing hemodialysis, and in patients with unstable angina. Aspirin is also effective in preventing stroke and death in patients with cerebral ischemia when administered in doses of approximately 1 gram per day. There are trends suggesting that aspirin is effective when administered in doses between 300 mg per day and 1500 mg per day in patients who have survived myocardial infarction. The side-effects of aspirin are mainly gastrointestinal and are dose-related. Generalized bleeding is very uncommon and limited mainly to patients with other hemostatic abnormalities or due to the concomitant use of anticoagulant therapy.

SummaryThe relative deficiency of antithrombin III (AT III) in neonatal plasma results in lower recovery of heparin in some assay systems. It is uncertain whether low AT III levels also limit the antithrombotic effects of heparin in this age group. We therefore compared the antithrombotic properties of heparin in mature pigs and newborn piglets, whose coagulation and inhibitor system closely resembles that of the human neonate. Animals were pretreated with saline, 10 or 25 U/kg heparin (n ≥16 per age group and dose). Following an injection of 100 U/kg thrombin, systemic 125I-fibrinogen consumption and local 125I-fibrinogen incorporation into jugular venous stasis thrombi were measured. Significantly more 125I-fibrinogen was consumed in piglets than in pigs systemically (p

Tissue-type plasminogen activator (t-PA) is a promising thrombolytic agent because it can produce thrombolysis without inducing a plasma proteolytic state. It is uncertain if this potentially important feature renders t-PA less hemorrhagic than other plasminogen activators. We have compared the hemorrhagic and thrombolytic effects of t-PA and streptokinase in rabbits. Streptokinase, 4000 U/kg/hr over 4 hr, failed to produce significant thrombolysis and 8000 U/kg/hr streptokinase over 4 hr produced only 28 +/- 6% thrombolysis. Both streptokinase regimens were associated with a plasmin-mediated plasma proteolytic state and both streptokinase regimens produced a significant increase in hemorrhage that was evident within 15 min of beginning the infusion and was progressive over the 4 hr of drug administration. In contrast, t-PA in a dose of 7500 U/kg/hr produced 35 +/- 6% thrombolysis, but it did not produce a plasmin-mediated plasma proteolytic state or a significant increase in hemorrhage over the 4 hr of infusion. t-PA in a dose of 15,000 U/kg/hr produced 85 +/- 4% thrombolysis but was associated with a plasmin-mediated proteolytic state and produced significant bleeding which, in contrast to streptokinase-induced bleeding, was delayed in onset. Therefore, t-PA induced less hemorrhage than streptokinase at doses that produced more effective thrombolysis. Bleeding with both thrombolytic agents was associated with a plasmin-mediated proteolytic state.

The newborn infant requires more heparin per kg body weight than the adult to achieve similar heparin plasma levels. Possible mechanisms include altered heparin pharmacokinetics and/or a decreased expression of anticoagulant activity of heparin in newborn plasma because of low levels of antithrombin III (AT-III). We measured the pharmacokinetics and the anticoagulant activity of heparin in the pig (AT-III level:100%), in the piglet (levels of AT-III: 50% of adult) and the piglet given exogenous porcine AT-III. All pigs were bolused with 125I-heparin (25 or 100 units/kg) and blood samples collected for the measurement of 125I-radioactivity, and antifactor Xa activity. The half-life of 125I-heparin was dose-dependent and similar in pigs and piglets; however, the volume of distribution was greater in the newborn resulting in an increased total clearance compared to the pig. The anti-factor Xa activity disappeared earlier in the piglet than in the pig. Both the kinetics and the absolute recovery of anti-factor Xa activity were normalized to pig values (after correction for different volumes of distribution) when the piglets were infused with exogenous AT-III. Thus apparent heparin resistance of the newborn is due to both an increased volume of distribution and the low AT-III level which limits the measurement of the anticoagulant activity of heparin in conventional anti-factor Xa assays.

SummaryTo test the possibility that different doses of heparin or other sulfated polysaccharides are required to inhibit thrombosis initiated by different stimuli, we compared the effects of heparin (HEP), pentosan polysulfate (SP54) and dermatan sulfate (DS) on the inhibition of thrombus formation induced by either I) tissue thromboplastin; II) thrombin; or III) factor Xa. Inhibition of thrombus formation induced by the stimuli was measured in a rabbit jugular vein hypercoagulation/stasis model. First, we determined the minimum dose of each sulfated polysaccharide which inhibited tissue thromboplastin-induced thrombus formation by ∼75%, and then compared the relative effectiveness of this dose to prevent thrombus formation initiated with the other two stimuli. HEP and SP54 were less effective when thrombin was the thrombogenic stimulus, while DS was more effective. HEP was the most effective agent when factor Xa was the stimulus. We conclude that the antithrombotic effectiveness of a given dose of a sulfated polysaccharide may vary depending on the stimulus which initiates thrombus formation.

There is now considerable evidence that the antithrombotic and the hemorrhagic effects of heparin can be dissociated by using low molecular weight heparins and by using heparin with low affinity to AT III. The findings with low-affinity heparin suggest that hemorrhage is contributed to by heparin properties that are independent of AT III binding and thus of their major anticoagulant effect. The current evidence supports the suggestion that the hemorrhagic effect of heparin is contributed to by a reversible platelet functional defect that is relatively less important than its AT III dependent anticoagulant effect for preventing experimental venous thrombosis. Whether these promising results in animals also apply to human thromboembolism disease will require careful evaluation by suitably designed clinical trials.

Standard heparin (SH) is frequently used in the sick neonate to prevent catheter related thrombosis. SH can cause significant bleeding complications in the adult and its use in the neonate is linked to an increased incidence of intraventricular hemorrhage. Recently available low molecular weight heparins (LMWH) offer potential advantages over SH in the adult by exhibiting a longer half life and decreased bleeding side effects compared to SH. Whether LMWHs would offer similar therapeutic advantages to the sick neonate is unknown. Using the porcine model of neonatal hemostasis we measured the pharmacokinetics of a LMWH (Choay 222) in the pig (ATIII level: 100%), in the piglet (ATIII level:50% of adult) and in the piglet given exogenous ATIII. All pigs were bolused with 125I-LMWH (5, 25 or 100 anti-factor Xa units/kg) and blood samples collected for the measurement of 125I-radioactivity, and anti-factor Xa activity. The half life of LMWH, measured as either 125I-radioactivity or as anti-factor Xa activity, was not dose dependent and was similar in pigs and piglets; however, the volume of distribution was greater in the piglet resulting in an increased total clearance compared to the pig. As well, the supplementation of the piglet with exogenous ATIII did not influence the pharmacokinetics of LMWH. The half life of the LMWH in both pigs and piglets was approximately twice as long as previously reported values for SH in the same animal model. Thus the longer half life of LMWH in the piglet, and the similarity of the half life in piglets and pigs suggest that LMWH may have a therapeutic advantage in the newborn over SH.

Summary. When 125I-fibrinogen scanning is used for the early detection of venous thrombosis, comparison with venography has shown a small percentage of abnormal scans associated with normal venograms. The mechanism of this discrepancy was investigated in an experimental model. Venous thrombosis was produced in 56 dogs using stasis and local thrombin. The data obtained from scanning and venography were validated by direct vein examination. 125I scanning agreed in 96% with venography when thrombus was present, but when thrombus lysed or embolized scans continued to read positive even though venograms were negative. Deliberate embolization of thrombi showed that these false positive scans continued for up to 48 hr owing to activity persisting in the vein wall. This was shown on histology to be related to fibrin deposition in the sub-endothelial and adventitial layers with inflammatory changes.

Clinical and experimental studies suggest that aspirin (ASA) is a useful antithrombotic agent; however, its efficacy seems limited to males. This may be due to differences in i) the effects of ASA on platelets and/or vessel wall or ii) the pharmacokinetics of ASA, between males and females.We examined the possible sex-related differences in 1) enhanced arterial thrombosis 3 h after treatment with 0, 3, 10 or 100 mg/kg ASA and 2) the distribution (α) and clearance (β) of ASA between male and female rabbits. Enhanced arterial thrombosis was determined by measuring 51Cr-platelet accumulation onto injured carotid arteries. ASA-plasma levels were determined, using a GC/MS assay. 51Cr-platelet accumulation onto injured arteries was 0.67 ± 0.15 × 106 (males) and 0.52 ± 0.07 × 106/100 mm2 vessel wall (females; mean ± SEM, n=8). 3 mg/kg ASA had no effect. 10 and 100 mg/kg ASA significantly enhanced 5lcr- platelet accumulation to 1.94 ± 0.41 × 106 and 2.59 ± 0.93 × 106 in males, and to 1.65 ± 0.32 × 106 and 2.16 ± 0.73 × 106 in females (pASA-plasma levels were determined in serial samples collected from 10 rabbits injected with 10 mg/kg ASA. One week later, the study was repeated in the same animals. The α and β T½’s were 3.3 ± 0.2 and 29.2 ± 3.0 (min) respectively in males after the 1st treatment. Similar T½’s were found after the 2nd treatment (3.4 ± 0.1, 23.3 ± 3.5 respectively). In females, the initial α and β T½’s were 3.7 ± 0.2 and 26.1 ± 3.5 respectively. The α T½ was similar (3.6 ± 0.1) whereas the β T½ was significantly decreased (14.8 ± 0.3, pThe significance of these findings is uncertain.

Methods are needed for screening patients at risk for thrombo-embolic diseases. We present an assessment of the relative usefulness of platelet shape distribution and mean volume measurements for probing abnormalities in ischemic heart disease (IHD). Platelet morphology in whole blood (WB) from the antecubital vein and/or abdominal aorta, and citrated platelet-rich plasma (PRP), which have been fixed by 4 volumes 0.8% glutaraldehyde-Tyrodes, pH 7.4, is compared for 17 healthy and 13 IHD donors: (I) 6 post-myocardial infarction (2-16 days), 1 acute coronary insufficiency (7 days) and (II) 6 angiographically characterized coronary artery disease. Platelets were examined under phase contrast microscopy classified as discocytes (D), disco-echi- nocytes (DE) and sphero-echinocytes (SE). WB of IHD donors has fewer D than WB from healthy donors (27(5-57)%* vs 71 (65-80%); *mean (range)) and more SE (24(6-65)% vs 1(0-8)%). Although platelet morphology in PRP from healthy donors Was similar to that of WB, PRP from TED donors contained fewer SE (3(0-11)%) and D (52(20-87)%) than WB. Platelet mean volumes, measured by microscopy, were increased by

Platelet survival (PS) may be decreased in some thromboembolic disorders. Treatment of patients with drugs that suppress platelet function may or may not be associated with normalisation of PS. It is uncertain, however, whether a lack of influence on PS necessarily indicates lack of an antithrombotic effect. We have examined this problem in an arterial thrombosis model in rabbits. A non-occlusive mural thrombus was produced in the aortic arch and PS studied using 51Cr-labelled homologous platelets. Thrombus size was assessed by measuring 51Cr accretion. Three groups of 15 animals were studied as follows:group A - thrombus induced 30 min prior to start of PS, group B- as for group A but animals treated with SUL (25 mg/Kg) and ASA (25 mg/Kg) prior to thrombus induction and 12 hrly thereafter and group C- sham operated controls. Animals in group B showed a 45% reduction in thrombus size when compared with group A (p

We have studied the prostaglandin I 2 production by endothelial and subendothelial cells of rabbit aortae and vena cavae. Rabbits were injected intravenously with 0, 10 or 100 mg/kg aspirin. One, 3, 6 or 30 hours later, segments of thoracic aorta or inferior vena cava were removed and their capacity to produce prostaglandin I 2 was measured, using a bioassay system. Both endothelial and subendothelial cells of the arteries and veins produced prostaglandin I 2 which was inhibited by aspirin in doses of 10 and 100 mg/kg. The inhibitory effect of aspirin on venous prostaglandin I 2 production was relatively short-lived and returned to baseline levels within 3 to 6 hours. In contrast, the inhibitory effect of aspirin on prostaglandin I 2 production by arteries lasted for at least 6 hours and by 20 hours had only returned to 50–70% of baseline. When experiments were performed in vitro, a similar difference in the duration of aspirin effect on venous and arterial prostaglandin I 2 production was also observed. These data are consistent with the hypothesis that the turnover of cyclo-oxygenase is more rapid in venous than in arterial cells.

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.

Bleeding is an important complication of heparin therapy. A number of low molecular weight heparin fractions produce less bleeding than standard heparin for an equivalent antithrombotic effect in experimental animals. Low molecular weight heparin fractions and fragments are produced by a number of different procedures but their relative effects on haemostasis and thrombosis have not been evaluated. We have compared the antithrombotic and haemorrhagic effects of two low molecular weight heparin fragments and of a heparinoid with porcine mucosa heparin and related these in vivo findings to the results of ex vivo tests of blood coagulation and in vitro tests of platelet vivo tests of blood coagulation and in vitro tests of platelet function. Haemorrhage was assessed using a rabbit ear bleeding model. The antithrombotic effects were assessed by measuring inhibition of a tissue thromboplastin-induced jugular vein thrombus and by inhibition of fibrin and platelet accumulation in an arterial-venous shunt. The ex vivo anticoagulant effects were assessed with the thrombin clotting time, activated partial thromboplastin time and anti-Xa assay, and the effect of these glycosaminoglycans on platelet function was assessed by measuring collagen-induced platelet aggregation. For a similar antithrombotic effect, standard heparin produced significantly more bleeding than the other 3 glycosaminoglycans. The antithrombotic effects of all 4 glycosaminoglycans occurred at similar levels of anti-Xa activity but there was no relationship between blood loss and the effects of these glycosaminoglycans on any of the other tests of blood coagulation. Standard heparin had a greater inhibitory effect on collagen-induced platelet aggregation than the low molecular weight glycosaminoglycans, supporting the possibility that the increased bleeding observed with heparin is related in part to its inhibitory effect on platelet function.

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.

In recent studies in rabbits, high dose aspirin (ASA) which inhibited prostacyclin(PGI2) synthesis by veins augmented thrombus formation in jugular veins. This thrombogenic effect was short-lived and lasted only 1-2 hrs. We have shown that the inhibitory effect of ASA on PGI2 synthesis in arteries lasts much longer than in veins. We therefore examined the effects of high dose ASA on arterial thrombosis in rabbits by measuring PGI2 production, 51Cr platelet adhesion and 125I-fibrin accretion onto a mechanically-induced injury site in carotid arteries. PGI2 production, quantitated by a thrombin-induced 14C-5HT release assay, was 0.13 ± 0.01 ng/10 mm2vessel. 51Cr platelet adhesion and 125I-fibrin accretion onto these vessels 1 hr after injury was 1.00 ± 0.13 × 106 platelets and 57 ± 5 μg fibrin/10 mm2 respectively (mean = SE, n = 22). After ASA treatment (100 mg/kg, IV), PGI2 production was inhibited by at least 84% for 6 hrs and returned to 56% of control by 20 hrs. This PGI2 inhibition was associated with a marked increase in platelet adhesion (>100%) which lasted for at least 20 hrs. There was no significant increase in fibrin accretion. These results show that the more prolonged effect of ASA on PGI2 production in arteries than in veins is paralleled by a more prolonged thrombogenic effect on arterial vessel wall injury.