Selective and sustained inhibition of surface-bound thrombin activity by intimatan/heparin cofactor II and its relevance to assessing systemic anticoagulation in vivo, ex vivo and in vitro.

We compare the relative activities of surface-bound and fluid-phase thrombin and their inhibition by heparin and Intimatan, a novel heparin cofactor II (HCII) agonist. In vitro, we compared the observed amidolytic activities of fluid-phase and surface-bound thrombin with the expected activities based upon 125I-specific activity. In vivo, we compared the inhibitory effects of heparin and Intimatan on thrombin activity bound to injured vessel walls. In vitro, the correlations between observed and expected activities of fluid-phase and surface-bound thrombin, were: r = 0.9974, p < 0.001; and r = 0.9678, p < 0.001; respectively. In vivo, injured vessel wall surface-bound thrombin activity persisted for > 24 h. This activity was not inhibited by heparin, but was inhibited by Intimatan, p < 0.001. We conclude that surface-bound thrombin is as active as fluid-phase thrombin and remains protected from inhibition by heparin, thereby contributing to vessel wall thrombogenicity following injury. In contrast, surface-bound thrombin is inhibited by Intimatan, thereby effectively decreasing vessel wall thrombogenicity following injury in vivo.

Results of the BRAT study--a pilot study investigating the possible significance of ASA nonresponsiveness on the benefits and risks of ASA on thrombosis in patients undergoing coronary artery bypass surgery.

BACKGROUND: Several studies suggest that acetylsalicylic acid (ASA) is less effective in preventing thrombotic events in ASA nonresponder patients. If so, the thrombotic event rate in ASA nonresponders should be higher than in ASA responders. OBJECTIVE: To conduct a prospective, multicentre observational pilot study to determine the thrombotic event rates in ASA responders and nonresponders. PATIENTS AND METHODS: Patients undergoing nonurgent coronary artery bypass grafting (CABG) who were prescribed 325 mg ASA/day were recruited. Patients were classified as an ASA responder or nonresponder based on the ASA effect (or lack thereof) on their bleeding times. All thrombotic events that occurred in the two years following CABG were recorded. These data were stored in a blinded fashion until the last patient follow-up, and then adjudicated by a validation committee. RESULTS: A total of 289 patients recruited at three sites completed the two-year follow-up. Of these patients, 45.3% were classified as ASA responders and 54.7% were classified as ASA nonresponders. Of ASA responders, 6.9% had thrombotic events compared with 9.5% of the ASA nonresponders, but this difference was not significant (P=0.526). CONCLUSIONS: While ASA responder or nonresponder status did not appear to affect the thrombotic event rate in patients undergoing nonurgent CABG, the possibility that ASA responder or nonresponder status affects the thrombotic event rate in more acutely ill CABG patients cannot be excluded.

Anticoagulant and antithrombin effects of intimatan, a heparin cofactor II agonist.

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.

A rationale for targeting antithrombotic therapy at the vessel wall: improved antithrombotic effect and decreased risk of bleeding.

Intimal hyperplasia after percutaneous transluminal coronary angioplasty (PTCA) or vascular surgical procedures remains a significant problem despite current antithrombotic therapy. The use of the current antithrombotic drugs, namely heparin + chronic aspirin (ASA) +/- oral anticoagulants, is based upon the assumptions that: i) heparin blocks thrombin generation and/or accelerates thrombin inhibition by antithrombin III (ATIII); ii) aspirin acetylates platelet cyclooxygenase, thereby preventing thromboxane A2 (TxA2) synthesis; and iii) oral anticoagulants reduce the availability of vitamin K-dependent procoagulants, thereby reducing the risk of thrombus formation. Albeit beneficial, this approach has a number of shortcomings and limitations: i) when thrombin binds to an injured vessel wall, it becomes resistant to inhibition by heparin/ATIII; thus, surface-bound thrombin remains active, stimulating further thrombus formation, smooth muscle cell proliferation and subsequent hyperplasia; ii) while TxA2 inhibition reduces platelet reactivity, platelets are able to respond to multiple stimuli generated at the time of, or after, vessel wall injury; and iii) heparin, aspirin and the oral anticoagulants all render the patient hemostatically defective and at risk of bleeding. Recent studies suggest that alternate therapeutic approaches can inhibit thrombogenesis more effectively at the time of injury, thereby not only inhibiting hyperplasia more effectively than the currently used drugs, but also reducing (or eliminating) the need for long-term therapy. For example, we suggest that the heparin cofactor II (HCII) catalysts, dermatan sulfate and Intimatan, inhibit surface-bound thrombin more effectively than heparin/ATIII, thereby inhibiting intimal hyperplasia effectively. Their effects are achieved when the drug is given only at the time of injury; i.e. with no further antithrombotic therapy. Other studies indicate that injured vessel wall thrombogenicity can be reduced by pretreatment with Persantine (dipyridamole) or with certain fatty acid supplements which either increase vessel wall cAMP and/or 13HODE synthesis. These increases are associated with decreased vessel wall thrombogenicity, which, in turn, is associated with decreased intimal hyperplasia. Such results suggest that vessel wall repair is achieved more effectively by targeting antithrombotic drugs directly at the vessel wall thrombogenicity per se rather than indirectly by altering the circulating blood cells and systemic coagulant system.

Cardiopulmonary bypass pumps and thrombin generation: what goes around comes around.

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 <50% of those seen in the RP pigs (p < 0.01 and p < 0.01, respectively). In Part 2, thrombin generation was significantly attenuated both during and after CPB in the CP patients (p < 0.01 and p < 0.01, respectively). However, there was no significant difference in fibrin deposition between the two types of pumps after their use in the patients undergoing cardiopulmonary bypass. It is concluded that there is less thrombin generation and subsequent fibrin deposition during CPB when using a CP instead of RP in a defined experimental in vivo situation, suggesting that there is less hypercoagulability during CPB when using a CP instead of an RP. However, a large study in more patients undergoing CPB for longer pump runs is required to determine the relevance of these observations on subsequent clinical endpoints.

Inhibition of chronic vessel wall intimal hyperplasia following acute anticoagulant treatment: relative effects of heparin and dermatan sulphate.

Surface-bound thrombin which contributes to vessel wall hyperplasia, is resistant to inhibition by heparin/antithrombin III (/ATIII) but not to inhibition by dermatan sulphate/heparin cofactor II (/HCII). To determine the effects of heparin and dermatan sulphate on vessel wall hyperplasia after a first or second injury, rabbit carotid arteries first were injured, using a standard procedure (first injury). Half of the first-injury rabbits were given heparin, dermatan sulphate, or saline, 5 minutes before and at 30-minute intervals over 2 hours post-injury, and then allowed to recover. Four weeks later, the first-injury treated animals were killed and their injured carotid arteries were processed histologically. The remaining untreated first-injury rabbits were also allowed to recover. At 4 weeks, those rabbits were re-anesthetized and their first-injury arteries (which were occluded >75%), were isolated, and vessel wall lumen patency was re-established by endarterectomy (second injury). During this second injury, the animals were treated with heparin, dermatan sulphate, or saline as described above. Four weeks after this second injury, these rabbits were killed and their second injury arteries were processed histologically. Intimal hyperplasia determined histologically, was expressed as an x-fold increase in vessel wall cross-sectional area (i.e., [(media+intima area) media area]). Vessel wall lumen occlusion was expressed as [1-(lumen area/internal elastic lamina area) x 100; % occlusion]. Vessel wall area in the saline-treated animals, increased 2.6+/-1.2 and 2.4+/-1.0 fold respectively, means+/-SD, n = 12, within 4 weeks of the first and second injuries. These increases were due to intimal hyperplasia and associated with 75+/-19% and 79+/-21% occlusion of the vessel wall lumen, respectively. Heparin had little effect, whereas dermatan sulphate (1) decreased hyperplasia by 45% after the first injury and by 47% after the second injury, p<0.008 and <0.03, respectively, and (2) decreased vessel wall occlusion 47+/-12% and 33+/-5% after the first and second injury, respectively. We conclude that (1) dermatan sulphate/HCII may be a useful inhibitor of vessel wall hyperplasia following vessel wall injury, and (2) this effect can be achieved by an acute anticoagulant treatment at the time of injury, unlike heparin/ATIII.

Regulation of endothelial cell and platelet receptor-ligand binding by the 12- and 15-lipoxygenase monohydroxides, 12-, 15-HETE and 13-HODE.

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.

Individual variation in the effects of ASA on platelet function: implications for the use of ASA clinically.

OBJECTIVE: To determine whether acetylsalicylic acid (ASA) inhibits hemostasis and platelet function in some individuals (ASA responders) but not in others (ASA nonresponders). DESIGN: In this two-part study, part 1 was a randomized, double-blind crossover study of the effects of various single doses of ASA (80 to 1300 mg) on primary hemostasis and platelet function. Part 2 was a prospective cohort study of the effects of a chronic dose of ASA (325 mg) on primary hemostasis and platelet function. SETTING: A hospital research laboratory and a cardiac care ward. SUBJECTS: Part 1: 10 healthy volunteers (five male, five female). Part 2: 40 consecutive patients undergoing elective coronary artery bypass grafting (CABG). RESULTS: Part 1: ASA, in a dose-related manner, prolonged the bleeding time in 60% of volunteers (ASA responders), which was associated with decreases in platelet thromboxane (Tx) A2 and 12-hydroxyeicosatetraenoic acid (12-HETE) synthesis and in platelet aggregation and adhesion. However, in volunteers whose bleeding time was not prolonged (ASA nonresponders), platelet 12-HETE synthesis and platelet adhesion were unchanged or increased (P < 0.001), despite platelet TxA2 and platelet aggregation being inhibited. Part 2: similarly, 58% of the CABG patients were ASA responders and all of their platelet biochemistry and function tests were inhibited, while in the CABG patient ASA nonresponders (no prolongation of bleeding time), platelet 12-HETE and platelet adhesion were increased (P < 0.001).

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

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

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

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

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

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

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

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

13-Hydroxyoctadecadienoic acid (13-HODE) metabolism and endothelial cell adhesion molecule expression: effect on platelet vessel wall adhesion.

Endothelial cells synthesize two important fatty acid metabolites, PGI2, which is synthesized from arachidonic acid via the cyclooxygenase pathway, and 13-HODE, which is synthesized from linoleic acid via the lipoxygenase pathway. PGI2 is synthesized following cell activation or injury while 13-HODE is synthesized in the unstimulated cell. While the role of PGI2 in platelet vessel wall interactions has been studied extensively, the role of 13-HODE in platelet vessel wall interactions is just now being understood. The present evidence suggests that 13-HODE is continuously synthesized in "resting" vessel wall cells and is in close juxtaposition with the ubiquous integrin adhesion molecule, the vitronectin receptor. The observation that the endothelial cell is not adhesive when 13-HODE and the vitronectin receptor are in close association and becomes adhesive when these two moieties dissociate and the vitronectin receptor relocates on the surface of the cell, provides further evidence that 13-HODE may induce conformational changes in the vitronectin receptor to reduce its ability to recognize its adhesive ligands. The additional observations that 13-HODE levels in both human and animal vessel walls are inversely related with vessel wall adhesivity, and that this adhesivity can be altered by altering 13-HODE synthesis, provides evidence that 13-HODE down-regulates the thrombogenecity of the injured vessel wall surface.