The first deletion mutation in the TSP1-6 repeat domain of ADAMTS13 in a family with inherited thrombotic thrombocytopenic purpura.

The inherited deficiency of ADAMTS13 is usually associated with severe forms of thrombotic thrombocytopenic purpura. Among the mutations identified in the ADAMTS13 gene, none have been described on the TSP1-6 repeat domain. We investigated an Iranian family with a history of chronic recurrent thrombotic thrombocytopenic purpura, severe ADAMTS13 deficiency and a heterogeneous pattern of clinical symptoms among affected members. Genetic analysis revealed a homozygous deletion of nucleotides 2930-2935 (GTGCCC) in exon 23 of ADAMTS13, leading to the replacement of Cys977 by a Trp and the deletion of Ala978 and Arg979 in the TSP1-6 repeat domain. To explore the mechanism of ADAMTS13 deficiency, in vitro expression studies were performed. Western blotting, pulse-chase labeling and immunofluorescence studies demonstrated a secretion pathway defect of the mutant protein, with no intracellular accumulation. This finding is consistent with the severe ADAMTS13 deficiency but does not explain the heterogeneous clinical picture of the 3 siblings carrying the same mutation.

Low vitamin B6 levels and the risk of recurrent venous thromboembolism.

Low plasma vitamin B6, measured as pyridoxal-5'-phosphate (PLP), is associated with an increased risk of first venous thromboembolism (VTE). In a prospective cohort of 757 patients with first VTE we investigated the association of PLP levels with risk of recurrent VTE. After 4 years, the likelihood of VTE recurrence among patients with PLP < or =23.3 nmol/L and 14.4% (11.5%-17.4%) among those with PLP >23.3 nmol/L was 22.5% (95% CI 13.6%-31.5%) (p=0.01). Patients with PLP ''23.3 nmol/L had 1.8-fold higher recurrence risk (1.01-3.14) than patients with PLP >23.3 nmol/L (adjusted for confounders including homocysteine). Therefore, low vitamin B6 is a risk factor of recurrent VTE.

Abnormalities of homocysteine and B vitamins in the nephrotic syndrome.

INTRODUCTION: The nephrotic syndrome is associated with heightened risk for arterial and venous thrombosis. Multiple derangements of hemostasis and acquired risk factors such as hyperlipidemia and hypertension contribute to this risk. The prevalence in the nephrotic syndrome of high circulating levels of homocysteine and of low levels of the B vitamins that are involved in its metabolism, which may play a role in thrombosis, is not well defined. MATERIALS AND METHODS: In 84 patients with nephrotic syndrome and 84 sex- and age-matched controls, hemostasis variables and the circulating levels of total homocysteine (tHcy), vitamin B(6), B(12) and folates were measured. RESULTS: tHcy levels were higher, vitamin B(6) and vitamin B(12) levels were lower in nephrotic patients than in controls. The association of low vitamin B(6) levels with the nephrotic syndrome was independent of any other alteration associated with the disease. Eighty-two percent of patients with the nephrotic syndrome had vitamin B(6) levels falling in the lowest quartile of the normal distribution. Antithrombin deficiency, factor V Leiden, antiphospholipid antibodies, hypertension, dyslipidemia, were more frequent in patients with the nephrotic syndrome than in controls. CONCLUSIONS: Patients with the nephrotic syndrome have multiple risk factors for thrombosis. We report that they frequently have low circulating levels of vitamin B(6), which associate with a heightened risk for venous and arterial thrombosis.

Both vitamin B6 and total homocysteine plasma levels predict long-term atherothrombotic events in healthy subjects.

AIMS: The contribution of homocysteine and group B vitamins in determining cardiovascular risk is debated. We assessed the predictive value of total homocysteine (tHcy), vitamin B12, folate, and vitamin B6 on the long-term occurrence of coronary and cerebral atherothrombotic events in a nested case-control study. METHODS AND RESULTS: Within a cohort of 1021 healthy subjects (490 men and 531 women) recruited in 1987, 66 first-ever coronary and 43 first-ever cerebrovascular events were recorded at a 12-year follow-up (cases, n=109). A total of 109 control subjects (remaining free from events) were matched with cases according to age, sex, smoking, hypertension, dyslipidaemia, and body mass index. Serum samples obtained in 1987 at baseline were used to measure tHcy, folate, and vitamins B12 and B6, as well as C-reactive protein plasma concentrations. We found a significant graded association between tHcy levels and the risk of coronary and cerebrovascular events [odds ratio (OR) for uppermost vs. lowermost quartile=1.34, 95% CI 1.01-1.76)]. Folate and vitamin B12 did not significantly differ between cases and controls, but were negatively (P<0.01) correlated with tHcy. Vitamin B6 did not correlate with tHcy levels, but differed significantly between cases and controls: for subjects in the uppermost quartile vs. the lowermost quartile of vitamin B6, OR=0.69 (95% CI 0.49-0.98). For subjects in the lowermost quartile of vitamin B6 and the uppermost quartile of tHcy, OR=17.50 (95% CI 1.97, 155.59). Cases and controls were not different as to C-reactive protein. CONCLUSION: tHcy and plasma vitamin B6 are long-term independent risk factors for coronary and cerebrovascular events.

Molecular mapping of the chloride-binding site in von Willebrand factor (VWF): energetics and conformational effects on the VWF/ADAMTS-13 interaction.

Physiological concentrations of NaCl inhibit the hydrolysis of von Willebrand factor (VWF) by ADAMTS-13. This effect is because of the specific binding of chloride ions to VWF. Urea-induced unfolding was measured in the presence of NaCl, CH3COONa, and NaClO4 at pH 8.0, 25 degrees C, for multimeric VWF, the recombinant A1-A2-A3 VWF domains, and the A1 domain. Chloride stabilizes the folded conformation of the A1-A2-A3 and A1 domains more efficiently than acetate but less strongly than perchlorate. Spectroscopic evidence showed that chloride binds to both the A1 and A1-A2 domain but not to the isolated A2 domain. Binding of Cl- to both wild type (WT) and the natural mutant p.R1306W A1-A2-A3 domains of VWF has a large heat capacity change equal to -1 and -0.4 kcal mol(-1) K(-1) for WT and p.R1306W A1-A2-A3 domains, respectively. This result implies that a burial of a vast apolar surface area is caused by conformational transitions linked to chloride binding. At any temperature, chloride affinity was higher for WT than for the mutant p.R1306W form. Chloride ions inhibit hydrolysis by ADAMTS-13 of the A1-A2-A3 and A1-A2 domains in the presence of either urea or high shear stress, whereas this effect was either absent or negligible in experiments using A2 and A2-A3 domains. These findings show that the A1 domain contains the binding site of chloride ions that control allosterically the proteolysis by ADAMTS-13 of the Tyr1605-Met1606 bond in the A2 domain and that the R1306W mutation of type 2B VWD quenches the binding of chloride ion to the A1 domain.

The thrombospondin-1 N700S polymorphism is associated with early myocardial infarction without altering von Willebrand factor multimer size.

The N700S polymorphism of thrombospondin-1 (TSP-1) has been identified as a potential genetic risk factor for myocardial infarction (MI). In a large case-control study of 1425 individuals who survived a myocardial infarction prior to age 45, the N700S polymorphism was a significant risk factor for myocardial infarction in both homozygous (odds ratio [OR] 1.9, 95% confidence interval [CI] 1.1-3.3, P = .01) and heterozygous carriers of the S700 allele (OR 1.4, 95% CI 1.1-3.3, P = .01). TSP-1 has been shown to reduce von Willebrand factor (VWF) multimer size, and the domain responsible for VWF-reducing activity has been localized to the calcium-binding C-terminal sequence. As the N700S polymorphism was previously shown to alter the function of this domain, we investigated whether the altered VWF-reducing activity of TSP-1 underlies the observed prothrombotic phenotype. The TSP1 N700S polymorphism did not influence VWF multimer size in patients homozygous for either allele nor was there a significant reduction of VWF multimer size following incubation with recombinant N700S fragments or platelet-derived TSP-1.

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 Tyr(1605)-Met(1606) 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 degrees C, with an apparent k(cat)/K(m) congruent with 3.4 x 10(4) M(-1) s(-1), but this value decreased by approximately 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 (ClO(4)(-) > Cl(-) > F(-)). 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 GpIbalpha 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.

Antiaggregatory activity in human platelets of potent antagonists of the P2Y 1 receptor.

Activation of the P2Y(1) nucleotide receptor in platelets by ADP causes changes in shape and aggregation, mediated by activation of phospholipase C (PLC). Recently, MRS2500(2-iodo-N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate) was introduced as a highly potent and selective antagonist for this receptor. We have studied the actions of MRS2500 in human platelets and compared these effects with the effects of two acyclic nucleotide analogues, a bisphosphate MRS2298 and a bisphosphonate derivative MRS2496, which act as P2Y(1) receptor antagonists, although less potently than MRS2500. Improved synthetic methods for MRS2500 and MRS2496 were devised. The bisphosphonate is predicted to be more stable in general in biological systems than phosphate antagonists due to the non-hydrolyzable CP bond. MRS2500 inhibited the ADP-induced aggregation of human platelets with an IC(50) value of 0.95 nM. MRS2298 and MRS2496 also both inhibited the ADP-induced aggregation of human platelets with IC(50) values of 62.8 nM and 1.5 microM, respectively. A similar order of potency was observed for the three antagonists in binding to the recombinant human P2Y(1) receptor and in inhibition of ADP-induced shape change and ADP-induced rise in intracellular Ca(2+). No substantial antagonism of the pathway linked to the inhibition of cyclic AMP was observed for the nucleotide derivatives, indicating no interaction of these three P2Y(1) receptor antagonists with the proaggregatory P2Y(12) receptor, which is also activated by ADP. Thus, all three of the bisphosphate derivatives are highly selective antagonists of the platelet P2Y(1) receptor, and MRS2500 is the most potent such antagonist yet reported.

Effects of some pre-analytical conditions on the measurement of homocysteine and cysteine in plasma.

The association of hyperhomocysteinemia and hypercysteinemia with the risk of arterial and venous thrombosis is well documented. While it is known that standardized pre-analytical conditions are necessary for reliable measurement of plasma total homocysteine, the effects of pre-analytical conditions on cysteine measurement are less well known. The aim of this study was to evaluate the effects of pre-analytical conditions on the measurement of homocysteine and cysteine. We observed that the concentration of total homocysteine in plasma increased significantly with time (38% after 6 h), whereas total cysteine decreased (5% after 2 h) when blood anticoagulated with ethylenediaminetetraacetic tripotassium salt was kept at room temperature. These changes were minimized when acidic citrate dextrose was used as an anticoagulant and were abolished when blood samples were immediately placed on crushed ice, independently of the anticoagulant. Storage of plasma for 72 h at room temperature induced a small (approximately equal to 6%), but significant, decrease in cysteine when blood was collected in ethylenediaminetetraacetic tripotassium salt. In contrast, homocysteine was stable in plasma for 72 h, independently of the anticoagulant used. In conclusion, if blood samples for plasma total homocysteine and cysteine measurement cannot be kept on ice, they should be collected in acidic citrate dextrose to minimize the artifactual changes.

Contribution of protease-activated receptors 1 and 4 and glycoprotein Ib-IX-V in the G(i)-independent activation of platelet Rap1B by thrombin.

Thrombin activates human platelets through three different membrane receptors, the protease-activated receptors PAR-1 and PAR-4 and the glycoprotein Ib (GPIb)-IX-V complex. We investigated the contribution of these three receptors to thrombin-induced activation of the small GTPase Rap1B. We found that, similarly to thrombin, selective stimulation of either PAR-1 or PAR-4 by specific activating peptides caused accumulation of GTP-bound Rap1B in a dose-dependent manner. By contrast, in PAR-1- and PAR-4-desensitized platelets, thrombin failed to activate Rap1B. Thrombin, PAR-1-, or PAR-4-activating peptides also induced the increase of intracellular Ca(2+) concentration and the release of serotonin in a dose-dependent manner. We found that activation of Rap1B by selected doses of agonists able to elicit comparable intracellular Ca(2+) increase and serotonin release was differently dependent on secreted ADP. In the presence of the ADP scavengers apyrase or phosphocreatine-phosphocreatine kinase, activation of Rap1B induced by stimulation of either PAR-1 or PAR-4 was totally inhibited. By contrast, thrombin-induced activation of Rap1B was only minimally affected by neutralization of secreted ADP. Concomitant stimulation of both PAR-1 and PAR-4 in the presence of ADP scavengers still resulted in a strongly reduced activation of Rap1B. A similar effect was also observed upon blockade of the P2Y12 receptor for ADP, as well as in P2Y12 receptor-deficient human platelets, but not after blockade of the P2Y1 receptor. Activation of Rap1B induced by thrombin was not affected by preincubation of platelets with the anti-GPIbalpha monoclonal antibody AK2 in the absence of ADP scavengers or a P2Y12 antagonist but was totally abolished when secreted ADP was neutralized or after blockade of the P2Y12 receptor. Similarly, cleavage of the extracellular portion of GPIbalpha by the cobra venom mocarhagin totally prevented Rap1B activation induced by thrombin in the presence of apyrase and in P2Y12 receptor-deficient platelets. By contrast, inhibition of MAP kinases or p160ROCK, which have been shown to be activated upon thrombin binding to GPIb-IX-V, did not affect agonist-induced activation of Rap1B in the presence of ADP scavengers. These results indicate that although both PAR-1 and PAR-4 signal Rap1B activation, the ability of thrombin to activate this GTPase independently of secreted ADP involves costimulation of both receptors as well as binding to GPIb-IX-V.

Measurement of warfarin in plasma by high performance liquid chromatography (HPLC) and its correlation with the international normalized ratio.

The measurement of plasma warfarin is required to investigate non-compliance, resistance to anticoagulation, drug metabolism and pharmacokinetic. Methods so far described are based on extraction of warfarin from plasma followed by reversed-phase HPLC. Extraction is the crucial step and may be performed in liquid- or solid-phase. The latter requires the preparation of columns, which makes the procedure variable. We investigated the suitability of the ready-for-use commercial cartridges for sample preparation. The method displayed between-run CV of 11.8%. Recovery was 99%. The coefficients of correlation between warfarin concentration in 50 patients and weekly dosage or INR were 0.55 (p<0.0001) or 0.25 (p=0.079).

[Primary angioplasty in acute myocardial infarction: experience and results in the first 1,000 consecutive patients]. / Angioplastica primaria nell'infarto micocardico acuto: esperienza e risultati nei primi 1000 pazienti consecutivi.

BACKGROUND: One of the biggest debates in modern cardiology regards the relative merit of primary percutaneous transluminal coronary angioplasty (PTCA) versus thrombolysis for the treatment of acute myocardial infarction with persistent ST-segment elevation. After the excellent results with primary PTCA in trials and meta-analyses, the next question is whether such results might be duplicated in "real world" conditions. METHODS: Between January 1995 and April 2003, 1000 consecutive patients with acute myocardial infarction, out of 2272 (44%) with ST-segment elevation admitted to the coronary care unit at the Cardiology Department of the S. Anna Hospital, were treated with PTCA. Our Institution is a medium-high volume center, without on-site surgery. Usual clinical and interventional practice, adjunctive antithrombotic therapy and results are described in this paper. RESULTS: Primary PTCA has been performed in 825 patients (75%) out of 1095 undergoing emergency angiography, "facilitated" in 140 (13%), rescue in 35 (3.2%). Eighty patients of the "facilitated" PTCA group had been pre-treated with tissue-type plasminogen activator 50 mg i.v. bolus, 50 with abciximab and 10 with reduced doses of fibrinolytic and abciximab. One hundred and seventy patients (16%) had been transferred to our Institution from community hospitals. Nine patients out of 1000 undergoing PTCA (0.9%) have been transferred immediately after the procedure (bail-out, failure) to perform urgent coronary artery bypass grafting. PTCA has been completed by stenting in 919 patients (92%). The median door-to-balloon time was 58 min (25th-75th percentile 49-71). The in-hospital total mortality rate was 4.9% (49 deaths): 5.3% (44 deaths) in the primary PTCA group, 2.1% (3 deaths) in the "facilitated" PTCA group (p = 0.042), and 5.7% (2 deaths) in the rescue PT-CA group. Early reinfarction rate was 1.5% (15 cases). The median time to hospital discharge was 10 days (25th-75th percentile 7-14). CONCLUSIONS: Since 9 years, our practice in the treatment of acute myocardial infarction with persistent ST-segment elevation is going on extending the use of primary PTCA, integrating pharmacological and mechanical options in selected cases.

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 microM ADP, similar to normal platelets with blocked P2Y(12) receptor. The response to 20 microM ADP, albeit still decreased, was more pronounced and was reduced by a P2Y(12) antagonist, indicating some residual receptor function. ADP failed to lower the adenylyl cyclase activity stimulated by prostaglandin E(1) in the patient's platelets, even though the number and affinity of 2-methylthioadenosine 5'-[(33)P]diphosphate-binding sites was normal. Analysis of the patient's P2Y(12) 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 P2Y(12) as compared with wild-type receptor. These studies delineate a region of P2Y(12) required for normal function after ADP binding.

Low vitamin B(6) 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 B(6) 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 B(6) 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 B(6) 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 B(6) levels lower than the 5th percentile of distribution in normal controls (p < 0.05). Low vitamin B(6) 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 B(6) 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 B(6) 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 alpha(1)-acid-glycoprotein (r = -0.37, 95% CI = -0.59 to -0.10, p < 0.01). Hyperhomocysteinemia was more frequent in patients with low vitamin B(6) 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 B(6) and homocysteine plasma levels (r = -0.13, 95% CI = -0.37 to +0.14, p = 0.33). CONCLUSIONS: Low vitamin B(6) plasma levels, an independent risk factor for thrombosis, are frequent in patients with IBD, especially those with active disease.