Refinement of the hereditary xerocytosis locus on chromosome 16q in a large Canadian kindred.

The hereditary stomatocytoses are a group of heterogeneous conditions associated with chronic red cell hemolysis for which the causative genetic mutations are not known. We investigated 137 members of a large Canadian kindred with phenotypic findings consistent with hereditary xerocytosis, one of the most common stomatocytosis syndromes. The objectives of this study were to characterize the clinical hallmarks of the hemolytic process, and to define the chromosomal region carrying the disease locus. The mode of inheritance was autosomal dominant. Affected family members had a well-compensated hemolysis, associated with an elevated MCHC, decreased osmotic fragility, decreased haptoglobin, and indirect hyperbilirubinemia. Cholelithiasis and progressive iron loading were common, despite normal hemoglobin levels. Quantitative erythrocyte morphologic evaluation revealed increased schistocytes, target cells, reticulocytes, and eccentrocytes in affected individuals; stomatocytes were not increased. Genetic linkage analysis confirmed the localization of the disease phenotype to chromosome 16q, and refined the candidate region to 16q24.2-16qter, a 2.4 million base pair interval containing 51 known or predicted genes.

Aggregating human platelets cause direct contraction and endothelium-dependent relaxation of isolated canine coronary arteries. Role of serotonin, thromboxane A2, and adenine nucleotides.

Aggregating human platelets contract isolated rings of canine coronary artery without endothelium, but relax rings with intact endothelium. We performed experiments to identify the substances released from platelets responsible for these effects. The contraction in rings without endothelium was reduced by treating the platelets with thromboxane synthetase inhibitor, dazoxiben, or treating the vessels with the thromboxane-receptor antagonist, SQ 29548. The serotonergic antagonist, methiothepin, also reduced the platelet-induced contraction. The combination of methiothepin plus dazoxiben or SQ 29548 caused a further inhibition. The endothelium-dependent relaxation to platelets during contractions evoked by prostaglandin F2 alpha was nearly abolished by the ADP- and ATP-scavenger, apyrase. It was not inhibited by methiothepin, which antagonizes endothelium-dependent relaxations to serotonin. Thus, both serotonin and thromboxane A2 contribute to the direct activation of coronary smooth muscle by aggregating human platelets, whereas adenine nucleotides are the principal mediators of the endothelium-dependent relaxation.

The influence of amines on various platelet responses.

Four amines, galactosamine, mannosamine, histamine and arginine were studied for their effects on platelet aggregation, platelet morphological changes, platelet protein phosphorylation and platelet secretion. Galactosamine inhibited platelet aggregation in response to arachidonic acid and ionophore A23187 but did not inhibit changes in platelet morphology, or in platelet protein phosphorylation in response to these agents and only partially inhibited platelet secretion. The results suggest that galactosamine can be used as a selective inhibitor of platelet-platelet attachment without having a significant effect on intracellular processes. Mannosamine was similar to galactosamine except that it partially suppressed phosphorylation of myosin light chain. Histamine was similar to mannosamine except that some platelet damage was seen in platelets exposed to histamine and arachidonic acid or ionophore A23187. Arginine was non-selective: it suppressed platelet aggregation, secretion and phosphorylation of myosin light chain and a 40 kDa protein (40P) in response to arachidonic acid and ionophore A23187. Arginine was also potent in suppressing platelet morphological changes. When the same four amines were evaluated for their effects on thrombin-induced aggregation; secretion was inhibited concomitantly with inhibition of aggregation. Inhibition of myosin light chain and 40P phosphorylation was evident with galactosamine, suggesting that when thrombin is used as the agonist, galactosamine is not a specific inhibitor of platelet-platelet attachment. These amines therefore have various effects on platelet responses. Under some conditions and with arachidonic acid or ionophore A23187 as agonist, one of them, galactosamine, can be used as a selective inhibitor of platelet-platelet attachment.

Use of recombinant factor VIIa (NovoSeven) in patients with Glanzmann thrombasthenia.

Recombinant factor VIIa (rFVIIa; NovoSeven, Novo Nordisk, Bagsvaerd, Denmark) appears effective and relatively safe for the treatment of bleeding and for surgical prophylaxis in patients with Glanzmann thrombasthenia as reported to the International Registry on rFVIIa and Congenital Platelet Disorders. One of the shortcomings of the Registry data is the heterogeneity of treatment protocol, including dosage, number of doses used, duration of treatment before declaration of failure, and mode of rFVIIa administration (bolus v continuous infusion). The data are not yet sufficient to define optimal regimens for various indications such as the type of bleeding or the type of procedures. The place of this drug compared to platelet transfusion in the overall management of patients with Glanzmann thrombasthenia will need to be determined in relationship to a number of challenges and unresolved issues in the clinical care of these patients. These issues include: how to improve local measures for patients with mucosal bleeds, optimal management of young women during menarche, optimal platelet transfusion regimens for various indications, the relationship between antiplatelet antibodies detected by monoclonal antibody-specific immobilization of platelet antigens (MAIPA) and effectiveness of platelet transfusion, whether there are other biological tests that may correlate with effectiveness of platelet transfusion, and management of pregnancy and delivery regarding antiplatelet immunization.

The incidence of deep venous thrombosis and pulmonary embolism among patients with inflammatory bowel disease: a population-based cohort study.

BACKGROUND: There is an impression mostly from specialty clinics that patients with inflammatory bowel disease (IBD) have an increased risk of venous thromboembolic disorders. Our aim was to determine the incidence of deep venous thrombosis (DVT) and pulmonary embolism (PE) from a population-based database of IBD patients and, to compare the incidence rates to that of an age, gender and geographically matched population control group. METHODS: IBD patients identified from the administrative claims data of the universal provincial insurance plan of Manitoba were matched 1:10 to randomly selected members of the general population without IBD by year, age, gender, and postal area of residence using Manitoba Health's population registry. The incidence of hospitalization for DVT and PE was calculated from hospital discharge abstracts using ICD-9-CM codes 451.1, 453.x for DVT and 415.1x for PE. Rates were calculated based on person-years of follow-up for 1984-1997. Comparisons to the population cohort yielded age-adjusted incidence rate ratios (IRR). Rates were calculated based on person-years of follow-up (Crohn's disease = 21,340, ulcerative colitis = 19,665) for 1984-1997. RESULTS: In Crohn's disease the incidence rate of DVT was 31.4/10,000 person-years and of PE was 10.3/10,000 person-years. In ulcerative colitis the incidence rates were 30.0/10,000 person-years for DVT and 19.8/10,000 person-years for PE. The IRR was 4.7 (95% CI, 3.5-6.3) for DVT and 2.9 (1.8-4.7) for PE in Crohn's disease and 2.8 (2.1-3.7) for DVT and 3.6 (2.5-5.2) for PE, in ulcerative colitis. There were no gender differences for IRR. The highest rates of DVT and PE were seen among patients over 60 years old; however the highest IRR for these events were among patients less than 40 years. CONCLUSION: IBD patients have a threefold increased risk of developing DVT or PE.

Serotonin and the vascular system. Role in health and disease, and implications for therapy.

Serotonin released from aggregating platelets can reach sufficient concentrations to affect local vascular function in a number of ways. The monoamine can cause contraction of blood vessels by its direct action on smooth muscle or by potentiating the effect of other vasoconstrictor agents. It can also induce vasodilatation by a direct relaxing effect on smooth muscle, by inhibition of adrenergic nerves, and by release of an uncharacterized relaxing factor from endothelial cells. One of its most likely physiological roles is to aid in haemostasis by promoting platelet aggregation and by causing local vasoconstriction at sites of injury. It probably has a role in some forms of vascular pathology as well: it may contribute to vasospasm of cerebral, coronary, and digital arteries, particularly if there is endothelial dysfunction or damage. Much evidence has implicated serotonin (5-hydroxytryptamine) in the pathogenesis of migraine. Serotonergic agonists, such as ergotamine, and antagonists, such as methysergide and pizotifen, are both used in therapy of migraine. Promising but conflicting early results have not yet defined a place for serotonergic antagonists in other vasospastic disorders. The antihypertensive efficacy of one serotonergic antagonist, ketanserin, raises questions about the possible involvement of serotonin in either the initiation or the maintenance of the elevated peripheral vascular resistance in several forms of hypertension, including essential hypertension.

Endothelium-dependent relaxations in human arteries.

Experiments were designed to study endothelium-dependent relaxations in human renal arteries (N = 13) and peripheral arteries (N = 8) suspended in organ chambers for isometric tension recording. In contracted arterial rings, acetylcholine caused endothelium-dependent relaxations that were not inhibited by indomethacin in either artery but were significantly augmented in the renal artery. Adenosine diphosphate and thrombin caused endothelium-dependent relaxations in renal but not in peripheral arteries. This finding suggests a heterogeneity of endothelium-dependent relaxations in human arteries and indicates that the relaxations are mediated by the release of an endothelium-derived relaxing factor (or factors) rather than the release of prostacyclin.

Differential effects of lower body negative pressure on forearm and calf blood flow.

Modest degrees of lower body negative pressure (less than 20 mmHg) cause a reflex constriction of forearm resistance vessels attributable to a decrease in activity of cardiopulmonary mechanoreceptors. In the present study, we sought to determine whether the calf vessels respond similarly. Left forearm and right calf blood flows were measured simultaneously by strain-gauge plethysmography in 10 healthy volunteers. Forearm flows decreased significantly from control during negative pressures of 10, 15, or 20 mmHg, whereas calf flows did not decrease significantly until 20 mmHg; at 10, 15, and 20 mmHg, decreases in forearm flow were significantly greater than those of the calf. Similar results were obtained in a second series of experiments in which venous pooling in the right leg during lower body negative pressure was prevented by enclosing it in a boot. At 40 mmHg, or after a Valsalva maneuver, both forearm and calf vessels constricted markedly and to the same degree. It appears that the reflex reduction in blood flow to the skeletal muscles of the limbs resulting from deactivation of the low-pressure intrathoracic mechanoreceptors is directed primarily to the arm.

Influence of endothelium-derived relaxing factor on platelet function and hemostasis in vivo.

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.

Osteonecrosis in a patient with Crohn's disease unrelated to corticosteroid use.

Osteonecrosis is a feared complication of corticosteroid use. However, a direct association between corticosteroid use and osteonecrosis has never been proven. The present report examines the case of a patient with longstanding Crohn's disease who had never been treated with corticosteroids and who developed osteonecrosis of the talus. The association of systemic inflammatory disorders with osteonecrosis and the possible association with vascular thrombosis are discussed.

Charcot foot.

Charcot joint is the painless, degenerative, progressive neuropathic destruction of the bony architecture of one or more joints of the feet. Diabetes mellitus is the most common cause of Charcot joint in North America, although the exact etiology is uncertain. The classic presenting complaint involves unilateral painless swelling of the lower extremity or foot. Charcot joint is often mistaken for cellulitis or deep vein thrombosis and may result in significant foot or ankle deformities. There are several treatment options for the patient presenting with Charcot joint. Medical management often includes immobilization and maintaining nonweightbearing status. Surgical intervention, often a final attempt at managing Charcot foot, involves careful patient selection and is not recommended for all patients. The postoperative phase can be challenging for both patient, nursing staff, and the surgeon.

Pathogenesis of Hyperhomocysteinemia-New Insights.

Mild to moderately elevated levels of homocysteine (Hey) in plasma, denoted as hyperhomocysteinemia, is emerging as a prevalent and strong risk factor for atherosclerotic vascular disease in coronary, cerebral and peripheral vessels, as well as for arterial and venous thromboembolism. Despite its clinical significance, the molecular mechanism of homocysteine's effects is not yet clearly defined. Most of the effects of homocysteine that have been demonstrated in vitro, affecting endothelial function have been attributed to the oxidant reactivity of this molecule, which is shown to affect the vasoregulatory and thrombotic/fibrinolytic function of endothelium. However, the relevance of these observations to the clinical situations is questionable, since excessively high concentrations of homocysteine are used in most of the experiments. We have observed that homocysteine, at physiologically relevant concentrations, specifically induces the expression of tissue factor by monocytes, and a non-specific redox effect is not involved. Tissue factor expression by monocytes is mediated by increased intracellular concentrations of the metabolic intermediate, S-adenosylhomocysteine, which is a potent inhibitor of methyl transferases. These studies suggest that tissue factor expression by circulating monocytes by intracellular perturbations may be a plausible mechanism by which homocysteine may induce thrombosis.

Comparison of serotonergic receptor subtypes on the smooth muscle and endothelium of the canine coronary artery.

To characterize the responses of the canine coronary artery to serotonin, rings with and without endothelium were suspended in organ chambers for isometric tension recording. Serotonin evoked an endothelium-dependent relaxation of prostaglandin F2 alpha-contracted rings which was inhibited by antagonists with affinity for 5-hydroxytryptamine (5-HT)1 and 5-HT2 receptors, methiothepin and metergoline, but was not mimicked or antagonized by the 5-HT1A-selective ligand, 8-hydroxy-2-di-n-propylamino tetralin. This relaxation is not mediated by 5-HT1B receptors as it was not antagonized by cyanopindolol; similarly, lack of inhibition by ketanserin and MDL 72222 rule out contributions of 5-HT2 receptors or 5-HT3 receptors. Rings without endothelium contracted to serotonin; this contraction was not blocked by cyanopindolol and was only weakly inhibited by ketanserin, but was antagonized in an apparently competitive fashion by methiothepin and was mimicked by 8-hydroxy-2-di-n-propylamino tetralin (although at higher concentrations than would be expected for its action at a 5-HT1A receptor). At high concentrations, serotonin evoked a relaxation of endothelium-denuded rings, which was blocked by very low concentrations of methiothepin but was unaffected by ketanserin or cyanopindolol. Thus, there appear to be three different serotonergic receptors in the coronary artery. Although available agents do not allow their precise classification as yet, none of them is of the 5-HT2 type.

Induction of monocyte tissue factor expression by homocysteine: a possible mechanism for thrombosis.

Moderately elevated plasma homocysteine levels are an important independent risk factor for arterial and venous thrombosis and for atherosclerosis. Some investigators have proposed that homocysteine's effects result from oxidant injury to the vascular endothelium or from an alteration in endothelial function. However, homocysteine may have other cellular targets. We now report that homocysteine, at physiologically relevant concentrations, induces the expression of tissue factor by monocytes. In response to homocysteine, monocytes express procoagulant activity in a dose-dependent and a time-dependent manner. This activity is attributable to tissue factor because it was dependent on factor VII and blocked by anti-tissue factor antibodies. Tissue factor mRNA levels were also increased in monocytes after homocysteine treatment. The effect was found to be specific because analogues of homocysteine (homocystine and homocysteine thiolactone) did not mimic homocysteine's activity, nor did other thiol compounds (cysteine, 2-mercaptoethanol, dithiothreitol). On the other hand, methionine, the metabolic precursor of homocysteine, was active though less potent than homocysteine. Catalase and superoxide dismutase (scavengers of H(2)O(2) and O(2)(-) Radicals, respectively) were unable to block the expression of tissue factor induced by homocysteine, as was a 5-fold excess of the reducing agent 2-mercaptoethanol. We conclude that the induction of tissue factor expression by circulating monocytes is a plausible mechanism by which homocysteine may induce thrombosis and that a nonspecific redox process is not involved.

Adenine nucleotides, serotonin, and endothelium-dependent relaxations to platelets.

Aggregating platelets cause an endothelium-dependent relaxation of isolated contracted canine coronary arteries. The role of adenine nucleotides and of 5-hydroxytryptamine in causing this relaxation was determined. Rings of these arteries were suspended in organ chambers filled with physiological salt solution and contracted with prostaglandin F2 alpha. Adenosine diphosphate relaxed rings with intact endothelium but had no effect on endothelium-denuded rings. The relaxation was attenuated by the enzyme, apyrase, which hydrolyzes adenosine tri- and diphosphate. 5-Hydroxytryptamine (5-HT) exerted a direct contractile effect mediated by the endothelium. The latter was prevented by the 5-HT1 serotonergic antagonist, methiothepin, but not by the 5-HT2 serotonergic antagonist, ketanserin. The endothelially mediated relaxation to aggregating platelets was prevented by apyrase but not by methiothepin or ketanserin. Responses to platelets were unaltered by the inhibitor of cyclooxygenase, meclofenamate. These experiments demonstrate the key role of adenine nucleotides in mediating the endothelium-dependent relaxation of canine coronary arteries to aggregating platelets.

Forearm vascular responses in normotensives and hypertensives after sublingual nifedipine.

Some hemodynamic variables of ten untreated hypertensives and nine normotensives were compared before and at 10, 30, and 60 min after nifedipine 10 mg sublingually. Serum nifedipine concentration was measured at each of these times, and was similar between groups. Resting forearm venous compliance did not differ between groups, and did not change after nifedipine. Nifedipine did not change mean arterial pressure significantly, but heart rate was increased in both groups. Resting forearm blood flow was significantly higher in hypertensives than normotensives and forearm vascular resistance was correspondingly lower. Forearm blood flow increased and vascular resistance fell after nifedipine in normotensives, but did not change significantly in hypertensives. Our data do not suggest any effect of nifedipine on peripheral venous compliance. We do not confirm the reported decrease in forearm venous compliance in hypertensives, but the characteristics of blood flow, vascular resistance, and lack of effect of nifedipine on blood pressure likely reflect a predominance of early hypertensives in our study population.

Endothelial cells and extracellular calmodulin inhibit monocyte tumor necrosis factor release and augment neutrophil elastase release.

Cultured human umbilical vein endothelial cells inhibited tumor necrosis factor-alpha release from whole blood or isolated mononuclear cells exposed to endotoxin. In contrast, the endothelial cells augmented neutrophil elastase release in the same blood. A protein with these functional properties was isolated from endothelial cell-conditioned media and, surprisingly, was identified as calmodulin. Authentic calmodulin mimicked the effect of endothelium. 125I-Calmodulin bound to a high affinity site on monocytic cell lines (Kd approximately 30 nM, in agreement with its functional activity). Cross-linking of 125I-calmodulin to monocytic cells identified a candidate calmodulin receptor. We conclude that calmodulin possesses an extracellular signaling role in addition to its intracellular regulatory functions. Calmodulin released at sites of tissue injury or possibly by specific mechanisms in the endothelium can bind to receptors, modulating the activities of inflammatory cells.

Inhibition of intravascular platelet aggregation by endothelium-derived relaxing factor: reversal by red blood cells.

Studies were performed to determine whether endothelium-derived relaxing factor (EDRF) can inhibit platelet aggregation within the vascular lumen, and if so, whether the inhibition persists in the presence of red blood cells (RBCs). Canine femoral arteries mounted in an organ bath were perfused with physiologic saline solution to which acetylsalicylic acid was added to block prostacyclin formation. During contraction with phenylephrine, addition of acetylcholine to the perfusing solution to evoke EDRF release relaxed the vessel wall. Washed human platelets labeled with 14C-5-hydroxytryptamine were added to the perfusing solution, and activated by thrombin infused via a branch vessel. The perfusate was collected downstream and centrifuged; the fraction of 14C-5-hydroxytryptamine appearing in the supernatant reflected the degree of platelet activation. Stimulation of EDRF release with acetylcholine inhibited 14C-5-hydroxytryptamine release. Hemoglobin (Hb) (10(-5) mol/L) blocked vascular relaxation and platelet-inhibition. RBCs at a hematocrit of 10% (treated with echothiophate to block erythrocyte cholinesterase) did not prevent relaxation but reversed the platelet inhibition. Lower hematocrits did not completely block the inhibition. Thus, erythrocyte Hb may modulate the inhibition of intraluminal platelet aggregation by EDRF.