Maddalena L. Zighetti

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.

A Common Mutation in the Methylenetetrahydrofolate Reductase Gene (C677T) Increases the Risk for Deep-Vein Thrombosis in Patients With Mutant Factor V (Factor V:Q506)

Hyperhomocysteinemia is a frequent risk factor for deep-vein thrombosis. A common mutation (C677T) in the gene encoding for methylenetetrahydrofolate reductase (MTHFR) is responsible, in the homozygous state, for decreased enzyme activity and mild hyperhomocysteinemia and is associated with increased risk for cardiovascular disease. We studied the prevalence of C677T MTHFR in 77 patients with deep-vein thrombosis and in 154 age- and sex-matched healthy control subjects. In the same individuals, we also evaluated the frequency of the coexistence of C677T MTHFR with mutant factor V:Q506, a common risk factor for deep-vein thrombosis. Sixteen patients (20.8%) and 35 control subjects (22.7%) were homozygous for the C677T MTHFR mutation (odds ratio [OR] = 0.8, 95% confidence interval [CI] = 0.4-2.0). Sixteen patients (20.8%) and 4 control subjects (2.6%) had factor V:Q506; of them, 10 patients and 3 control subjects had isolated factor V:Q506 (adjusted OR = 6.3, 95% CI = 1.6-25.3) and 6 patients and 1 control subject also had C677T MTHFR (adjusted OR = 17.3, 95% CI = 2.0-152.9). The OR for the coexistence of the two mutations was 65% to 75% higher than the expected joint effect calculated by either an additive (OR = 6.0) or multiplicative (OR = 4.4) model. The homozygous C677T mutation of MTHFR per se is not a risk factor for deep-vein thrombosis but increases the risk associated with factor V:Q506. Due to the high prevalence of C677T MTHFR, it is likely that previous studies, which did not look for this mutation, overestimated the relative risk of thrombosis associated with factor V:Q506 alone.

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.

Role of ADP in platelet aggregation at high shear: studies in a patient with congenital defect of platelet responses to ADP.

Summary. The in vitro measurement of platelet aggregation (PA) at the high shear levels that can be found in the microcirculation may provide useful informations on primary haemostasis, which is usually explored in vivo with the skin bleeding time (BT). PA at high shear requires von Willebrand factor (vWf) and the platelet glycoprotein (GP) complexes Ib/IX/V and IIb/IIIa; controversial results have been reported on its requirement of released adenosine diphosphate (ADP). Due to its dependence on vWf, PA at high shear may be affected by the vasopressin analogue DDAVP, which increases the plasma vWf levels and shortens the prolonged BT of patients with congenital or acquired defects of platelet function. We studied PA at high shear, BT and plasma vWf levels in a patient with congenital impairment of platelet responses to ADP before and after the i.v. infusion of 0.3 μg/kg DDAVP. Two methods to study PA at high shear were used: shear‐induced PA (SIPA) and the filter aggregation test. With both methods, PA at high shear of the patient was impaired. The infusion of DDAVP increased plasma vWf levels, shortened the prolonged BT and potentiated PA at high shear of the patient. In conclusion, PA at high shear is impaired in a patient with congenital defect of platelet responses to ADP and prolonged BT and is potentiated by DDAVP. Our results suggest that released ADP plays an important role in PA at high shear and that potentiation of PA at high shear by DDAVP may be one mechanism by which the drug shortens the prolonged BT of patients with congenital or acquired defects of platelet function.

Absence of hyperhomocysteinemia in ten patients with primary pulmonary hypertension

Primary pulmonary hypertension is a disease of unknown etiology, characterised by the elevation of pulmonary arterial pressure eventually leading to right ventricular failure and death (1). Although the pathogenetic mechanisms are heterogeneous (1,2), evidence from histopathological studies suggests that a primary thrombotic process may be responsible for the disease in most patients (3, 4). Among the conditions predisposing to thrombosis is congenital homocystinuria (5), which, in a case report, was shown to be associated with acute cor pulmonale due to the formation of thrombi in pulmonary vessels (6). Since also moderate hyperhomocysteinemia is associated with increased risk for early onset venous and arterial thrombosis (7-9), we measured its prevalence in 10 patients with primary pulmonary hypertension.

Plasma Levels of Activated Protein C in Healthy Subjects and Patients With Previous Venous Thromboembolism Relationships With Plasma Homocysteine Levels

The proteolytic enzyme activated protein C (APC) is a normal plasma component, indicating that protein C (PC) is continuously activated in vivo. High concentrations of homocysteine (Hcy) inhibit the activation of PC in vitro; this effect may account for the high risk for thrombosis in patients with hyperhomocysteinemia (HyperHcy). We measured the plasma levels of APC in 128 patients with previous venous thromboembolism (VTE) and in 98 age- and sex-matched healthy controls and correlated them with the plasma levels of total Hcy (tHcy) measured before and after an oral methionine loading (PML). Forty-eight patients had HyperHcy and 80 had normal levels of tHcy. No subject was known to have any of the congenital or acquired thrombophilic states at the time of the study. Because the plasma levels of APC and PC were correlated in healthy controls, the APC/PC ratios were also analyzed. Plasma APC levels and APC/PC ratios were significantly higher in VTE patients than in controls (P=0.03 and 0.0004, respectively). Most of the increase in APC levels and APC/PC ratios were attributable to patients with HyperHcy. Patients with normal tHcy had intermediate values, which did not differ significantly from those of healthy controls. There was no correlation between the plasma levels of tHcy or its PML increments and APC or APC/PC ratios in controls. The fasting plasma levels of APC and APC/PC ratios of 10 controls did not increase 4 hours PML, despite a 2-fold increase in tHcy. This study indicates that APC plasma levels are sensitive markers of activation of the hemostatic system in vivo and that Hcy does not interfere with the activation of PC in vivo.

Usefulness of a flow cytometric analysis of intraplatelet vasodilator-stimulated phosphoprotein phosphorylation for the detection of patients with genetic defects of the platelet P2Y(12) receptor for ADP.

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

Abstract 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 2h) 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 72h at room temperature induced a small (≅6%), but significant, decrease in cysteine when blood was collected in ethylenediaminetetraacetic tripotassium salt. In contrast, homocysteine was stable in plasma for 72h, 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.

Low risk of thrombosis in family members of patients with hyperhomocysteinaemia.

Summary. Mild to moderate hyperhomocysteinaemia, a metabolic disorder due to genetic and/or acquired factors, is associated with an increased risk of venous and arterial thrombosis. To establish whether measuring homocysteine in members of families of hyperhomocysteinaemic patients is warranted, we investigated 169 relatives of patients diagnosed with hyperhomocysteinaemia after they developed arterial or venous thrombosis. The prevalence of hyperhomocysteinaemia was 16·6%; the relative risk of thrombosis in relatives with hyperhomocysteinaemia compared to those without was 1·2 (odds ratio; 95% CI 0·24–4·2), with similarly low absolute annual incidences of thrombosis (0·28% and 0·24%). The low prevalence of hyperhomocysteinaemia among relatives of patients with this metabolic disorder, and their low risk of thrombosis, do not justify family screening.

Partial inhibition of platelet aggregation by nebulized pentamidine in severe haemophiliacs

The antiparasite agent pentamidine has been shown to inhibit human platelet aggregation in vitro at concentrations that (potentially) may be attained in patient plasma after the administration of the drug by nebulizer. We measured platelet aggregation in platelet‐rich plasma (PRP) before and after the administration of 300 mg nebulized pentamidine to 10 HIV‐positive patients with severe haemophilia on prophylaxis against Pneumocystis carinii pneumonia. All patients had normal platelet counts. PAF‐acether, U46619, collagen and ADP at different concentrations were used as agonists. Platelet aggregation was lower in PRP samples taken at the end of pentamidine administration and 1 h thereafter than in samples taken at the same time points in control experiments (without the administration of pentamidine). The inhibition of platelet aggregation was mild and tended to be overcome by higher concentrations of platelet agonists. The bleeding time was prolonged from 5 to 15 min in one patient but did not change in the remaining nine patients. In conclusion, this controlled study shows that nebulized pentamidine inhibits platelet aggregation in HIV‐positive haemophiliacs without significantly affecting their bleeding times. Although this mild inhibitory effect may not be clinically relevant in haemophiliacs with normal platelet counts despite their defect in intrinsic coagulation, patients with HIV‐related thrombocytopenia should be monitored to detect any excessive prolongation of their bleeding times after nebulized pentamidine.