Rossana Lombardi

Deamino-8-D-arginine vasopressin shortens the bleeding time in uremia

In a randomized double-blind cross-over trial we gave either 1-deamino-8-D-arginine vasopressin or placebo to 12 patients with uremia, hemorrhagic tendencies, and prolonged bleeding times. After vasopressin infusion, all patients had shortened bleeding times, with the effect lasting for at least four hours in most cases. Platelet count, platelet cyclic AMP levels, platelet retention on glass beads, plasma fibronectin, serum thromboxane B2 and residual prothrombin, hematocrit, and plasma osmolarity were unchanged after vasopressin. A consistent post-infusion increase in factor VIII coagulant activity and, to a lesser extent, in factor VIII-related antigen and ristocetin cofactor accompanied the shortening of bleeding time. In addition, vasopressin induced the appearance in plasma of larger von Willebrand-factor multimers than those present in the resting state. The compound was given to nine additional patients with acute or chronic renal failure and prolonged bleeding times, before major surgery or renal biopsy. In these patients, shortening of the bleeding time was associated with normal hemostasis. Our findings indicate that 1-deamino-8-D-arginine vasopressin can be used for temporary correction of bleeding time and may prevent surgical bleeding in patients with uremia.

Conjugated estrogens for the management of bleeding associated with renal failure.

Bleeding is a major complication of uremia. Both cryoprecipitate and desmopressin effectively shorten the prolonged bleeding time and favorably influence clinical bleeding, but the former carries the risk of transmitting blood-borne infectious diseases, and both cryoprecipitate and desmopressin have a short duration of action. Preliminary evidence has suggested that estrogens may be useful, and we therefore performed a randomized, double-blind, crossover trial comparing the effect of conjugated estrogens with that of placebo on hemorrhagic tendencies and the bleeding time in six patients with uremia who were on maintenance hemodialysis. Five daily infusions of placebo or conjugated estrogens were administered at the beginning of one-month trial periods. Estrogen shortened the bleeding time in all six patients. The effect was detectable six hours after the first infusion, reached its maximum in all patients between days 5 and 7, and lasted for 14 days. By day 16 after the last infusion, the bleeding time had returned to base line in four of the six patients. No side effects were noted during or after estrogen infusion. Estrogens did not influence the circulating level of von Willebrand factor or change its multimeric structure. Moreover, the defective platelet aggregation and thromboxane formation observed in the patients were not corrected by estrogens. We conclude that conjugated estrogens are an adequate alternative to cryoprecipitate or desmopressin for the treatment of bleeding associated with renal failure, especially when a longer duration of action is needed and immediate onset of the effect is not essential. The mechanism of action of estrogens remains to be clarified.

Molecular bases of defective signal transduction in the platelet P2Y12 receptor of a patient with congenital bleeding

We have identified structural attributes required for signal transduction through a seven-transmembrane-domain receptor. Platelets from a patient (AC) with a congenital bleeding disorder had normal shape change but reduced and reversible aggregation in response to 4 μM ADP, similar to normal platelets with blocked P2Y12 receptor. The response to 20 μM ADP, albeit still decreased, was more pronounced and was reduced by a P2Y12 antagonist, indicating some residual receptor function. ADP failed to lower the adenylyl cyclase activity stimulated by prostaglandin E1 in the patients platelets, even though the number and affinity of 2-methylthioadenosine 5′-[33P]diphosphate-binding sites was normal. Analysis of the patients P2Y12 gene revealed a G-to-A transition in one allele, changing the codon for Arg-256 in the sixth transmembrane domain to Gln, and a C-to-T transition in the other allele, changing the codon for Arg-265 in the third extracellular loop to Trp. Neither mutation interfered with receptor surface expression but both altered function, since ADP inhibited the forskolin-induced increase of cAMP markedly less in cells transfected with either mutant P2Y12 as compared with wild-type receptor. These studies delineate a region of P2Y12 required for normal function after ADP binding.

Low vitamin B6 plasma levels, a risk factor for thrombosis, in inflammatory bowel disease: Role of inflammation and correlation with acute phase reactants

OBJECTIVES:Individuals with inflammatory bowel disease (IBD) are at increased risk for thrombosis and vitamin deficiencies. Low plasma levels of vitamin B6 are an independent risk factor for thrombosis and may cause hyperhomocysteinemia, another recognized risk factor for thrombosis. The aim of this study was to evaluate vitamin B6 plasma levels in IBD patients.METHODS:We studied 61 IBD patients: 32 with Crohn’s disease and 29 with ulcerative colitis. For each patient, three sex- and age-matched healthy control subjects were studied.RESULTS:Median vitamin B6 levels were significantly lower in IBD patients (22.0 pmol/L, range 3.6–231.0) than in controls (31.1 pmol/L, 3.7–363.4; p < 0.01). In all, 13.1% IBD patients and 5.5% controls had plasma vitamin B6 levels lower than the 5th percentile of distribution in normal controls (p < 0.05). Low vitamin B6 levels were significantly more frequent in patients with active disease than in patients with quiescent disease (seven of 26, 26.9%, vs one of 35, 2.9%; p < 0.001). Moreover, patients with active disease had significantly lower median vitamin B6 levels (13.4 pmol/L, range 3.6–124.0) than patients in a quiescent phase (27.0 pmol/L, 7.8–231.0; p < 0.001). Low vitamin B6 levels were significantly correlated with serum concentrations of C-reactive protein (r = −0.36, 95% CI = −0.59 to −0.09, p < 0.01) and α1-acid-glycoprotein (r = −0.37, 95% CI = −0.59 to −0.10, p < 0.01). Hyperhomocysteinemia was more frequent in patients with low vitamin B6 levels (three of eight, 37.5%) than in patients with normal levels (nine of 53, 17.0%; p = 0.18). There was no statistically significant correlation between vitamin B6 and homocysteine plasma levels (r = −0.13, 95% CI = −0.37 to +0.14, p = 0.33).CONCLUSIONS:Low vitamin B6 plasma levels, an independent risk factor for thrombosis, are frequent in patients with IBD, especially those with active disease.

Aberrant multimeric structure of von Willebrand factor in a new variant of von Willebrand's disease (type IIC).

A variant of von Willebrands disease has been identified in which sodium dodecyl sulfate agarose electrophoresis provides evidence that the von Willebrand factor present is structurally abnormal. Rather than the repeating triplet seen in normal subjects and in patients with the IIA and IIB variants, a repeating doublet was present in the propositus. None of the bands had the same mobility as bands in normal subjects or previously described von Willebrands disease patients. The larger multimers of von Willebrand factor were lacking both from plasma and platelets, and did not appear in the circulation after infusion of 1-deamino-[8-D-arginine]-vasopressin. There was a marked increase in the concentration of the smallest multimer in the propositus and his phenotypically normal children, indicating that this abnormality of von Willebrand factor is inherited in an autosomal-recessive manner.

Conversion of recent onset atrial fibrillation to sinus rhythm using a single oral loading dose of propafenone: comparison of two regimens

A population of 105 patients with recent onset (< 72 h) atrial fibrillation was randomly treated with propafenone as a single oral loading dose of 450 mg (Regimen A) or 600 mg (Regimen B) or with placebo. A 24-h Holter was performed. Criteria of efficacy were conversion to sinus rhythm at 2, 4 and 8 h compared to placebo and also significant reduction of mean ventricular rate in persistent atrial fibrillation. After 2 h, regimen B was more effective than either regimen A (43% vs. 8%; p = 0.001) or placebo (11%; p = 0.004). At 4 h, both the active treatments were more effective than placebo (17% vs. 46% regimen A and 57% vs. regimen B; p < 0.04 and p < 0.001, respectively). Sinus rhythm resumed within 24 h in 71%, 80% and 69% of the patients with regimen A, B and placebo, respectively (p = not significant). The mean ventricular rate reduction after 1 h was 8%, 11% and 4% for regimen A, B and placebo, respectively (p < 0.005 vs. regimen B), and 17%, 25% and 6% respectively (p < 0.001 placebo vs. regimen A and B, p < 0.05 regimen B vs. A) at 2 h. No major adverse effect occurred. Atrial flutter with 1:1 atrioventricular conduction only in one case who received placebo. Propafenone acute oral administration is more effective than placebo in rapidly converting recent-onset atrial fibrillation to sinus rhythm and may be the treatment of choice in this setting limiting hospitalization and contributing to improved quality of life.

Shear-induced platelet aggregation is potentiated by desmopressin and inhibited by ticlopidine.

Shear-induced platelet aggregation is important in physiological hemostasis and in the pathogenesis of arterial thrombosis. It requires extracellular Ca2+, platelet membrane glycoproteins Ib/IX and IIb/IIIa, von Willebrand factor (vWF), and ADP. We studied the effects of desmopressin (DDAVP), which increases plasma vWF levels and shortens the bleeding time, and of ticlopidine, which inhibits platelet responses to ADP, on shear-induced platelet aggregation. Eleven healthy volunteers were given oral ticlopidine (250 mg b.i.d.) for 7 days. The same subjects were infused intravenously with DDAVP (0.3 micrograms/kg body wt) before the first and after the last doses of ticlopidine. The degree of platelet aggregation induced by shear stress at 25, 50, 75, and 100 dyne/cm2 in a cone-and-plate viscometer, plasma vWF levels, and the bleeding time were measured before and after each DDAVP infusion. Plasma vWF levels and the extent of shear-induced platelet aggregation increased after DDAVP and were correlated. Ticlopidine partially inhibited shear-induced platelet aggregation both before and after DDAVP infusion. The bleeding time, prolonged by ticlopidine, was shortened by DDAVP. Potentiation by DDAVP of shear-induced platelet aggregation may be one mechanism by which the drug shortens the prolonged bleeding time. Since shear-induced platelet aggregation can cause thrombotic occlusions in stenotic arterial vessels, our findings may explain the therapeutic efficacy of ticlopidine in arterial thrombosis.

Regular ventricular rhythms before conversion of recent onset atrial fibrillation to sinus rhythm.

The incidence of fast atrial tachycardias with regular ventricular rhythm was assessed in a population of 243 patients with recent onset (< 72 hours) atrial fibrillation (AF), without heart failure, randomly treated with single loading oral dose of propafenone (600 mg), flecainide (300 mg), digoxin (1 mg), or placebo for acute conversion to sinus rhythm (SR). Fast atrial arrhythmias developed in 14 (6%) patients: 6/92 treated with propafenone, 3/34 treated with flecainide, 1/25 treated with digoxin, and 4/ 92 who received placebo (P = NS). Heart rate > 175 beats/min with 1:1 AV conduction ensued in 4 cases: 2 treated with flecainide and 2 treated with placebo; in the other cases 2:1 AV conduction was observed. Widening of QRS during regular tachycardia was observed in 4 patients; 3 who received propafenone and 1 who received flecainide. Conversion to SR within 4 hours was achieved in 55/92 (60%) patients treated with propafenone, 20/34 (59%) patients treated with flecainide, 7/25 (28%) patients treated with digoxin, and 19/92 (20%) treated with placebo (P < 0.001 propafenone vs placebo and flecainide vs placebo; P < 0.05 propafenone vs digoxin and flecainide vs digoxin). Periods of regular tachycardia are expected in recent onset AF and may not necessarily represent a proarrhythmic effect of Class 1C drugs, rather than mark the transition from AF to SR. Class 1C agents are probably responsible for widening of the QRS complex seen during these tachycardias. Propafenone and flecainide appear equally effective in converting recent onset AF.

Alterations of Factor VIII von Willebrand Factor in Clinical Conditions Associated with an Increase in its Plasma Concentration

Summary. Factor VIII‐related antigen (VIIIR: Ag) was consistently higher than factor‐VIII procoagulant activity (VIII:C) in 57 patients with clinical conditions characterized by acute‐phase reactions. Two different methods for measuring VIII:C (one‐ and two‐stage assays) and VIIIR: Ag (electroimmunodiffusion and immunoradiometric assay) gave concordant results in the majority of cases. In 43% of plasma samples, crossed immunoelectrophoresis in agarose gel was characterized by the appearance of an additional, fast‐moving precipitin peak which was immunologically identical with the major, slower‐moving VIIIR : Ag peak. The fast‐moving peak was detected in all the patients with clinical conditions typically associated with increased plasma proteolysis (DIC, acute pancreatitis, during thrombolytic therapy). It was present in a smaller proportion of cases with liver and renal failure and malignancies and in the post‐operative period. The additional VIIIR : Ag peak is thought to be the result of in vivo factor VIII/von Willebrand factor fragmentation by proteolytic enzymes.

Role of Chloride Ions in Modulation of the Interaction between von Willebrand Factor and ADAMTS-13

The degradation of von Willebrand factor (VWF) depends on the activity of a zinc protease (referred to as ADAMTS-13), which cleaves VWF at the Tyr1605-Met1606 peptide bond. Little information is available on the physiological mechanisms involved in regulation of AD-AMTS-13 activity. In this study, the role of ions on the ADAMTS-13/VWF interaction was investigated. In the presence of 1.5 m urea, the protease cleaved multimeric VWF in the absence of NaCl at pH 8.00 and 37 °C, with an apparent kcat/Km ≅ 3.4 × 104 m-1 s-1, but this value decreased by ∼10-fold in the presence of 0.15 m NaCl. Using several monovalent salts, the inhibitory effect was attributed mostly to anions, whose potency was inversely related to the corresponding Jones-Dole viscosity B coefficients (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{ClO}_{4}^{-}{>}\mathrm{Cl}^{-}{>}\mathrm{F}^{-}\) \end{document}). The specific inhibitory effect of anions was due to their binding to VWF, which caused a conformational change responsible for quenching the intrinsic fluorescence of the protein and reducing tyrosine exposition to bulk solvent. Ristocetin binding to VWF could reduce the apparent affinity and reverse the inhibitory effect of chloride. We hypothesize that, after secretion into the extracellular compartment, VWF is bound by chloride ions abundantly present in this milieu, becoming unavailable to proteolysis by AD-AMTS-13. Shear forces, which facilitate GpIbα binding (this effect being artificially obtained by ristocetin), can reverse the inhibitory effect of chloride, whose concentration gradient across the cell membrane may represent a simple but efficient strategy to regulate the enzymatic activity of ADAMTS-13.