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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.

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.

A non-specific cation channel in cultured human umbilical vein endothelial cells was obtained by cell-attached patch-clamp study. This channel showed a conductance of 28 pS when both pipette and bath contained 140 mM potassium chloride. when pipette solution was changed into 140 sodium chloride with 5 mM calcium chloride, the conductance was 26 pS. when 120 mM calcium chloride was used as the only cation in the pipette, a conductance of 6 pS was obtained. Bath application of cyclopiazonic acid, an inhibitor of the sarcoplasmic reticulum Ca2+ pump in smooth muscle and other tissues, dose dependently activates this non-specific cation channel. It is assumed that cyclopiazonic acid by blockade of the refilling of Ca2+ stores depletes the rapidly exchanging intracellular Ca2+ stores and this action stimulates Ca2+ influx through the non-specific cation channels in human umbilical vein endothelial cells.

HE INTACT endothelial cell (EC) lining of the vascuT lar wall is not reactive to the circulating blood However, ECs acquire thrombogenic properties when they are stimulated by cytokines or thrombin, as shown both in vivo and in vitro. Thus, there is expression of plasminogen activator inhibit~r,~ expression of tissue fa~tor,~ and downregulation of thrombomod~lin,~ all of which can facilitate thrombogenesis. We have made a number of observations that suggest that changes in EC lipoxygenase metabolism after stimulation also influence the thrombogenic properties of ECs. First, we found that unstimulated ECs metabolize linoleic acid into 13-hydroxyoctadecadienoic acid (1 3HODE) via the lipoxygenase pathway, the intracellular levels of which correlate inversely with EC thrombogenecity.6 Second, we and others have reported that, when ECs are stimulated by thrombin or by any of a number of cytokines, 13-HODE synthesis is inhibited and EC reactivity to platelets and cancer cells increases, both in vitro and in Finally, recent studies indicate that the increased EC reactivity is associated with an enhanced expression of the vitronectin receptor (VnR) on ihe apical surface of the ECs.I6,l7 Therefore, we hypothesized that these two phenomena are related and constitute an important mechanism by which EC reactivity for platelets, cancer cells, and other circulating blood cells is regulated. One possible mechanism by which 13-HODE may influence VnR expression is that 13-HODE may interact with the VnR, altering its conformational presentation and, therefore, its ability to recognize its specific ligands. Consistent with that possibility, Conforti et all8 found that the ability of the VnR to recognize von Willebrand factor (vWF), fibronectin, and vitronectin was dependent, in part, on the plasma membrane fatty acid(s) with which it associated. Other investigators have also reported that various fatty acids derived from activated platelets and polymorphonuclear leukocytes enhance the abilities of other argineglycine-aspartic acid (RGD)-recognizing integrins to bind their ligands in purified sy~tems.’~,~~ Thus, a number of investigators have provided evidence that lipid moieties may be important in modulating the ligand-binding properties of integrins. The studies cited above raise the possibility that

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 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 effects of human recombinant interleukin-1 alpha and beta (rIL-1 alpha; rIL-1 beta) on the adhesion of human A549 lung carcinoma cells and M6 melanoma cells (TC) to human endothelial cells (HECs) in vitro were studied, and on TC/lung entrapment in vivo. In vitro, there was a significant increase in TC/HEC adhesion to HECs pretreated for 4 h with rIL-1 alpha or rIL-1 beta. The effects of rIL-1 alpha and beta on TC/HEC adhesion were time dependent and reached a plateau within 4-6 h. TC/HEC adhesion was not blocked when measured in the presence of antibodies to either fibronectin, glycoprotein IIb/IIIa, anti-ICAM, or anti-LFA. However, enhanced TC/HEC adhesion was completely blocked in the presence of the peptide, GRGDS. In vivo, pretreatment of nude mice for 4 h with rIL-1 alpha (given i.p. before i.v. injection of TCs) enhanced TC retention in the lung 24 h later. Our data demonstrate that IL-1 enhances TC adhesion to the vascular surface both in vitro and in vivo, suggesting that IL-1 can facilitate the metastatic process.

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.

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.

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.

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.

Depletion of internal Ca 2+ stores causes capacitative Ca 2+ entry which occurs through non-selective cation channels sensitive to blockade by SK&F 96365. Recently, alkaloids of Chinese herbal medicinal origin, tetrandrine and hernandezine, have been shown to possess actions including inhibition of Ca 2+ channels in non-excitable cell types. In this study, we compared the actions of these novel inhibitors to those of SK&F 96365 in fura-2-loaded endothelial cells from human umbilical vein and bovine pulmonary artery. Depletion of Ca 2+ from the internal stores was accomplished in Ca 2+ -free medium using an endoplasmic reticulum Ca 2+ pump inhibitor, cyclopiazonic acid (CPA) or receptor agonists, histamine and bradykinin. Stimulation with histamine or bradykinin caused a marked and rapid transient increase in Ca 2+ signal whereas CPA caused a smaller amplitude increase of longer duration. Restoring Ca 2+ to the medium caused marked and sustained increases in the fluorescence indicating movement of Ca 2+ into the cytosol presumably stimulated by the emptied Ca 2+ stores. SK&F 96365 as well as tetrandrine and hernandezine antagonized depletion-induced Ca 2+ entry. The results suggest that these putative inhibitors interact with Ca 2+ entry triggered by depletion of the internal Ca 2+ stores and their action is presumed to be on the non-selective cation channels. Their effectiveness may be enhanced by the mechanisms which lead to the opening of the Ca 2+ influx channel.

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.

Tumor cells (TC)/vascular endothelial cell (EC) adhesion is important in metastasis. In vitro studies indicate that the EC linoleic acid (18:2) derived monohydroxide, 13-HODE 1/αwith TC/EC adhesion and with EC expression of the vitronectin receptor (VnR). In addition, the ability of tumor cells to adhere 1/α with its intracellular ratio of 13-HODE and the arachidonic acid derived monohydroxide, 15- hydroxyeicosatetraenoic acid (15-HETE). These data suggest that changing TC or EC 13-HODE synthesis will alter TC/EC interactions and subsequent metastasis in vivo Consistent with this possibility, we found that the abilities of TCs to arrest and metastasize in the rodent lung 1/αwith the ratio of TC 13- HODE: 15-HETE. We also found that when vascular wall (VW) 13-HODE synthesis is inhibited, acute TC/lung entrapment and chronic TC metastasis increase, p

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.

The relative thrombogenicity of extracellular matrixes (ECMs) produced by cultured human umbilical endothelial cells (ECs) was studied under flow conditions. ECMs were prepared using a number of physical and chemical methods, and their reactivity toward platelets was morphometrically evaluated. von Willebrand factor (vWF), fibronectin (FN), and 13-hydroxy-9-cis,11-trans-octadecadienoic acid (13-HODE) were also determined. We found that platelet adhesion to ECMs differed significantly, both quantitatively and qualitatively, with the method of ECM preparation. Mechanically prepared ECM exposed a less thrombogenic surface compared with ECM prepared by chemical methods (platelet-covered surface of 20% and 50%, respectively). Evaluation of the ECM components vWF, FN, and 13-HODE showed significant changes, both in their concentrations and distribution patterns, depending on the method of ECM preparation. The decrease measured in the levels of ECM-associated vWF (from 108 to 9.2 ng/10(4) cells) and the minor changes observed in the distribution pattern of subendothelial FN did not appear to be sufficient to explain the altered platelet adhesion observed in our model. This suggests that the amount of 13-HODE probably associated to the remaining ECs present in the mechanically exposed ECM could be one factor that specifically contributed to the nonthrombogenic state of these preparations. We conclude that the degree of ECM reactivity toward platelets is dependent on the method of ECM preparation and that this is related to the removal of specific EC/ECM components that modulate their thromboresistant/thrombogenic properties. This fact should be taken into account when ECMs produced by cultured ECs are used in platelet adhesion studies.

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.

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.

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

SummaryPrevious studies have demonstrated that standard anticoagulant tests are poor indices of the antithrombotic potential of glycosaminoglycans which are weak catalysts of the thrombinantithrombin III reaction. In this study we investigated whether the catalysis of thrombin inhibition by plasma could serve as a reliable index for assessing the antithrombotic effectiveness of glycosaminoglycans. Equal volumes of 125I-thrombin and control or test plasma were incubated for up to 10 min at 37° C. Inactivation of thrombin was then determined after 7.9% SDS-polyacrylamide gel electrophoresis and subsequent autoradiography. Increasing concentrations of heparin (>0.066 μg/mL or 0.01 USP units/mL) and dermatan sulfate (>0.1 μg/mL) could be readily demonstrated in undiluted plasma by enhanced formation of complexes of thrombin with antithrombin III and heparin cofactor II respectively. However, the detection of any catalytic effect of the two glycosaminoglycans decreased significantly with increasing plasma dilutions. When ex vivo plasmas obtained from rabbits that had been injected with the minimum dose of any one of seven glycosaminoglycans required to achieve their optimal antithrombotic effect were assessed for their ability to catalyse thrombin inhibition, there was approximately a 2-fold increase in the amount of thrombin inactivated 30 s after the thrombin had been added to the plasma. The enhanced inhibition of thrombin was achieved by catalysis of antithrombin III and/or heparin cofactor II activities. These results suggest that measurement of the catalysis of thrombin inactivation in undiluted plasma is a sensitive and reliable index for estimating the antithrombotic potential of glycosaminoglycans in rabbits.

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

This work demonstrates that after bolus intravenous injection standard heparin (SH) disappearance results from the combination of a saturable and a non saturable mechanism. Pharmacokinetics and pharmacodynamics of SH were studied by measuring the disappearance of increasing doses (5 - 500 anti-factor Xa U/kg) of 125I-heparin and of its biological effects. CPM curves allowed to determine the half lives of heparin according to the dose injected. The half lives were clearly dose dependent and reached a plateau over 100 anti-factor Xa U/kg. The complex curve which describes the amount of heparin cleared per time unit after any given dose has been resolved into its two components reflecting a saturable and a non saturable mechanism of disappearance. For the doses less than 100 anti-factor Xa U/kg the saturable mechanism was preeminent and the anti-factor Xa activity disappearance followed an exponential pattern; for the doses less than 100 anti-factor Xa U/kg the contribution of the non saturable mechanism becomes more important and the anti-factor Xa activity disappearance followed a concave-convex pattern. Further experiments showed that the heparin half life shortened as the circulating anti-factor Xa activity decreased; this phenomenon may explain the concave-convex pattern of the curve of the anticoagulant effect observed after injection of large doses of SH.

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

Polymorphonuclear leukocytes (PMN) adhere to the vascular endothelial lining in vivo and to the surfaces of cultured endothelial cells in vitro, but the mechanisms of these cellular interactions remain unclear. Arachidonic acid metabolites, both cyclooxygenase- and lipoxygenase-derived, have been shown to influence PMN locomotion, secretion, and adhesion to artificial surfaces. To determine whether such mediators also are involved in regulating PMN-endothelial cell interactions, we have examined the effects of prostacyclin and various inhibitors of arachidonic acid metabolism on the adherence of radiolabeled PMN to cultured bovine aortic endothelial cells. Confluent endothelial monolayers were incubated with washed suspensions of radiolabeled human PMN (which contained less than 1% platelet contamination) at 37 degrees C for 30 min, then subjected to a standardized wash procedure and the number of adherent leukocytes determined radiometrically. Under basal conditions, i.e., in the absence of exogenous activating stimuli, 4,163 +/- 545 PMN adhered per square millimeter of endothelial surface (mean +/- SEM, n = 12). This basal adhesion (which corresponds to approximately 4–5 leukocytes per endothelial cell) was unaffected when the leukocytes and endothelial monolayers were pretreated with cyclooxygenase inhibitors (100 microM aspirin or 1–5 microM indomethacin) or PGI2 (10(-9)-10(6) M). Thus, basal PMN-endothelial adhesion in this in vitro model system does not appear to be dependent on endogenous cyclooxygenase derivatives of arachidonate or to be sensitive to inhibition by exogenous prostacyclin. In contrast, leukocyte adhesion was significantly reduced by pretreatment with 5,8,11,14- or 4,7,10,13-eicosatetraynoic acid, 0.5- 5 mM sodium salicylate, or 10–1,000 microM indomethacin, antiinflammatory agents that can interfere with the metabolism of arachidonic acid via non-cyclooxygenase-dependent mechanisms. These observations may be relevant to the interactions of circulating PMN with vascular endothelium under both physiologic and pathophysiologic conditions in vivo.

Fatty acid metabolism by vascular endothelial cells occurs both under basal conditions and following endothelial cell stimulation or injury. Under basal conditions, endothelial cells are metabolically very active and rapidly turn over triglycerides and synthesize 13-hydroxyoctadecadienoic acid from linoleic acid via the cytosolic enzyme, hω6-lipoxygenase. When endothelial cells are stimulated (or injured), these pathways are turned off, and, instead, arachidonic acid is liberated from the membrane phospholipids and metabolized into prostacyclin via the cyclooxygenase enzyme. The biological relevance of these two fatty acid metabolites is discussed in respect to the regulation of cell/cell interactions during both ‘homeostasis’ and ‘injury’.

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

We performed studies to examine the mechanism responsible for the antithrombogenic effect of aspirin in experimental animals. We measured the effect of 10 and 100 mg/kg doses of aspirin, salicylate, or a combination of both on the accumulation of radiolabeled platelets onto injured carotid arteries in rabbits. The effects of these agents on thrombogenicity measured as platelet accumulation onto injured carotid arteries, were correlated with the ability to inhibit platelet thromboxane A2 and vessel wall prostacyclin synthesis. We found that a low dose of aspirin significantly inhibited platelet accumulation onto the injured vessels, while a high dose reversed this effect. Thromboxane A2 and prostacyclin production, however, were maximally inhibited in rabbits given either aspirin dose. The reversal of the antithrombotic effect of the low dose of aspirin by a high dose of aspirin was simulated by administering a combination dose of high-dose salicylate and low-dose aspirin. We concluded that the reversal of the antithrombotic effect of a 10 mg/kg dose of aspirin by a higher dose of aspirin could not be explained solely by the inhibition of PGI2 synthesis and was affected by the salicylate moiety of aspirin.

Summary The effects of standard heparin, three low molecular weight derivatives of heparin, dermatan sulphate and pentosan polysulphate on the intrinsic coagulation pathway were compared in order to evaluate the contributions of the anti-factor Xa and anti-thrombin activities to their anticoagulant activities. The anticoagulant potency was measured by the ability of each sulphated polysaccharide to inhibit the generation of thrombin activity in plasma. Similarly, the ability of the six sulphated polysaccharides to enhance the rates of inactivation either factor Xa or thrombin in defibrinated plasma containing calcium chloride and cephalin were also determined. Standard heparin was the only sulphated polysaccharide that could equally inhibit thrombin generation and enhance the inactivation of factor Xa and thrombin by plasma. Dermatan sulphate and pentosan polysulphate were more effective as inhibitors of thrombin generation than potentiators of factor Xa inactivation. The two smallest derivatives of heparin, which had high anti-factor Xa (but low antithrombin) activity, were the poorest inhibitors of thrombin generation. Our results therefore suggest that only sulphated polysaccharides that enhance the inactivation of thrombin by plasma and/or inhibit the generation of thrombin activity in plasma are good anticoagulants. These two activities of sulphated polysaccharides appear to be good predictors of the relative antithrombotic potency in vivo.

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.

A number of methods have been used to measure various lipoxygenase metabolites in aqueous samples. These methods, however, suffer from three major limitations: first, they require extensive extraction and isolation from protein-containing media; second, mainly due to the first limitation, they have poor recoveries; and third, these methods usually require a two-step procedure, one for the actual extraction and the other for the quantification of the lipoxygenase metabolites. We have developed a fully automated high-performance liquid chromatographic method which circumvents these limitations. As a result, we are able to obtain high recoveries of various lipoxygenase metabolites from protein-containing samples (i.e. biological samples) while simultaneously quantifying each metabolite. The method employs a column venting technique, whereby the fatty acids are extracted by a pre-column and the proteins are vented to waste. The pre-column eluate is then directed through the analytical column which separates the lipoxygenase metabolites. The described method is reproducible and minimizes both the time and the cost involved in assaying a sample.

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

The relationship between two anticoagulant actions of glycosaminoglycans (GAGs), namely the catalysis of thrombin inhibition (assessed by thrombin-antithrombin-III and thrombin-heparin-cofactor-II formation) and the inhibition of prothrombin activation, was explored by comparing the effects of heparin, heparan sulfate, and dermatan sulfate on the two reactions in plasma. Heparan sulfate and dermatan sulfate were also resulfated in vitro to yield products with sulfate/carboxylate ratios similar to those of heparin. Their effects on thrombin inhibition and the activation of prothrombin were also determined. The catalytic efficiency of the five GAGs on thrombin inhibition and their inhibitory effects on prothrombin activation decreased in the following order: heparin; resulfated dermatan sulfate; resulfated heparan sulfate; heparan sulfate = dermatan sulfate. These results suggest that the catalytic efficiency of a glycosaminoglycan on thrombin inhibition translates to its inhibitory effect on prothrombin activation, since catalysis of thrombin inhibition results in the inhibition of the thrombin-dependent positive feedback reactions of coagulation which facilitate prothrombinase formation.

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