Anna Lecchi

Purinoceptors on blood platelets: further pharmacological and clinical evidence to suggest the presence of two ADP receptors

Summary. Platelet aggregation by ADP plays a major role in the development and extension of arterial thrombosis. The antithrombotic thienopyridine compounds ticlopidine and clopidogrel have proved useful tools to investigate the mechanisms of ADP‐induced platelet activation. In essence, although clopidogrel has been shown to completely and selectively block ADP‐induced platelet aggregation, G protein activation and inhibition of adenylyl cyclase, this drug does not affect shape change and Ca2+ influx. Binding studies, using the non‐ hydrolysable ligand [33P]2MeSADP, have shown that human platelets contain about 600 high‐affinity binding sites for 2MeSADP (Kd∼ 5 niw). These sites present pharmacological characteristics of a P2T receptor. Clopidogrel treatment reduces the number of sites by 70% on rat platelets (from 1200 to 450) and leaves the residual binding sites resistant to clopidogrel. Moreover, patients with congenital impairment of ADP‐induced platelet aggregation but normal shape change display very low levels of [33P]2MeSADP binding sites.The current data thus strongly suggest the presence of two ADP receptors, one responsible for shape change and rapid Ca2+ influx and the other a Gi protein‐coupled receptor responsible for Ca2+ mobilization from internal stores, inhibition of adenylyl cyclase and platelet aggregation.

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.

Evaluation of Platelet Function with the PFA-100 System in Patients with Congenital Defects of Platelet Secretion

The template bleeding time is still the screening test for defects of platelet function, although it is an invasive and poorly reproducible technique. The PFA-100 measures platelet function at high shear. Whole blood is aspirated through a capillary to an aperture of a membrane coated with platelet agonists. The system measures the time required to obtain occlusion of the aperture by a platelet plug (closure time). We measured the closure times in the PFA-100 system and the bleeding time in seven patients with delta-storage pool deficiency, 10 patients with primary secretion defect (not due to abnormalities of platelet granules or the arachidonate pathway), and 40 controls. Measurements were repeated I and 4 hours after intravenous infusion of desmopressin in six delta-storage pool deficiency and eight primary secretion defect patients. Baseline bleeding time and closure times with the collagen/epinephrine cartridge were longer in delta-storage pool deficiency and primary secretion defect patients than in controls. In contrast, closure times with the collagen/adenosine diphosphate cartridge were normal in both delta-storage pool deficiency and primary secretion defect patients. Treatment with desmopressin increased the plasma von Willebrand Factor levels, shortened the prolonged bleeding time, shortened the closure times with the collagen/adenosine diphosphate cartridge, and normalized the closure times with the collagen/ epinephrine cartridge. Therefore, the PFA-100 test may be a less invasive alternative to the bleeding time in the diagnosis and therapeutic monitoring of patients with platelet secretion defects. The collagen/epinephrine cartridge is more sensitive than the collagen/adenosine diphosphate cartridge to defects of platelet secretion.

Usefulness of PFA‐100® testing in the diagnostic screening of patients with suspected abnormalities of hemostasis: comparison with the bleeding time

Summary.u2002 Background:u2002Global tests of hemostasis that are used to screen patients with clinical suspicion of bleeding disorders should help the physician to identify the phase of the hemostatic system that is abnormal and guide further diagnostic workup.

Prevalence of hyperhomocysteinemia in adult gluten-sensitive enteropathy at diagnosis: Role of B12, folate, and genetics

BACKGROUND & AIMSnHyperhomocysteinemia, a risk factor for thrombosis, recurrent miscarriages, and osteoporosis, might derive from acquired folate and vitamin B 12 deficiencies and from a C677T mutation in methylene-tetrahydrofolate reductase (MTHFR) gene. Undiagnosed gluten-sensitive enteropathy (GSE) is associated with vitamin deficiencies, osteoporosis, and recurrent miscarriages. We evaluated the prevalence and the risk factors for hyperhomocysteinemia in patients with newly diagnosed GSE.nnnMETHODSnIn this prospective study performed in a tertiary care setting, 40 consecutive subjects with newly diagnosed GSE were evaluated for homocysteine, folate, and vitamin B 12 levels and for C677T polymorphism. One hundred twenty sex- and age-matched healthy control subjects were studied. Nonparametric tests and multiple regression analysis were used to evaluate the risk factors in inducing hyperhomocysteinemia in the GSE population.nnnRESULTSnHyperhomocysteinemia was more frequent in GSE patients than in control subjects (8/40, 20.0% vs 7/120, 5.8%) (relative risk, 3.4; 95% confidence interval, 1.3-8.9), as well as folate deficiency (17/40, 42.5% vs 10/120, 8.3%) (relative risk, 5.1; 95% confidence interval, 2.5-10.2). Multiple regression analysis showed that folate and B 12 levels were independently and inversely associated with homocysteine levels, whereas homozygosity for the MTHFR thermolabile variant was not. The prevalence of MTHFR variant in GSE population was not different from that reported in racially comparable control groups. Gluten-free diet was able to normalize folate, vitamin B 12 , and homocysteine levels.nnnCONCLUSIONSnHyperhomocysteinemia is frequent in newly diagnosed GSE. Vitamin deficiencies caused by malabsorption are the most important determinants of this condition. Hyperhomocysteinemia might contribute to the occurrence of common complications of undiagnosed GSE.

Inhibition of the platelet P2Y12 receptor for adenosine diphosphate potentiates the antiplatelet effect of prostacyclin

Background:u2002Activation of two receptors for adenosine diphosphate (ADP), P2Y1 and P2Y12, is necessary for ADP‐induced platelet aggregation (PA). It is generally believed that the antithrombotic effects of drugs inhibiting P2Y12, such as clopidogrel, are uniquely mediated by inhibition of P2Y12‐dependent PA. However, as P2Y12 is negatively coupled to adenylyl cyclase (AC), its inhibition may also exert antithrombotic effects through the potentiation of prostacyclin (PGI2), which inhibit PA by stimulating AC. Objectives:u2002To test whether inhibition of P2Y12 potentiates the antiplatelet effects of PGI2. Methods:u2002We measured the effects of PGI2 (0.01–10u2003μm) on PA of washed human platelets induced by thrombin (0.5u2003Uu2003mL−1) in the presence or absence of ARC69931MX (anti‐P2Y12) or MRS2500 (anti‐P2Y1). Results:u2002PGI2 inhibited PA in the presence of anti‐P2Y12, but not in the presence of anti‐P2Y1 or in the absence of inhibitors. In contrast, dibutyryl‐cyclicAMP inhibited PA both in the presence and absence of anti‐P2Y1 or anti‐P2Y12. PGI2 increased platelet cyclicAMP levels only in the absence of thrombin or in the presence of thrombin plus anti‐P2Y12. Conclusions:u2002PGI2 did not inhibit PA induced by thrombin, because its effect on AC was prevented by released ADP interacting with P2Y12. Anti‐P2Y12 drugs, by rescuing AC activity, potentiate the antiplatelet effect of PGI2, which may contribute to their antithrombotic effect.

Rapid stimulation of tyrosine phosphorylation signals downstream of G-protein-coupled receptors for thromboxane A2 in human platelets

Signals ensuing from trimeric G-protein-coupled receptors synergize to induce platelet activation. At low doses, the thromboxane A2 analogue U46619 does not activate integrin alphaIIbbeta3 or trigger platelet aggregation, but it induces shape changes. In the present study, we addressed whether low doses of U46619 trigger tyrosine phosphorylation independently of integrin alphaIIbbeta3 activation and ADP secretion, and synergize with adrenaline (epinephrine) to induce aggregation in acetylsalicylic acid (aspirin)-treated platelets. Low doses of U46619 triggered tyrosine phosphorylation of different proteins, including FAK (focal adhesion kinase), Src and Syk, independently of signals ensuing from integrin alphaIIbbeta3 or ADP receptors engaged by secreted ADP. The G(12/13)-mediated Rho/Rho-kinase pathway was also increased by low doses of U46619; however, this pathway was not upstream of tyrosine phosphorylation, because this occurred in the presence of the Rho-kinase inhibitor Y-27632. Although low doses of U46619 or adrenaline alone were unable to trigger platelet aggregation and integrin alphaIIbbeta3 activation, the combination of the two stimuli effectively induced these responses. PP2, a tyrosine kinase inhibitor, and Y-27632 inhibited platelet activation induced by low doses of U46619 plus adrenaline and, when used in combination, totally suppressed this platelet response. In addition, the two inhibitors selectively blocked tyrosine kinases and the Rho/Rho-kinase pathway respectively. These findings suggest that both tyrosine phosphorylation and the Rho/Rho-kinase pathway are required to activate platelet aggregation via G(12/13) plus G(z) signalling.

Role of MRP4 (ABCC4) in Platelet Adenine Nucleotide-Storage : Evidence from Patients with Delta-Storage Pool Deficiencies

We previously showed that the MRP4 (ABCC4) transporter is expressed in human platelet delta-granules and may be involved in ADP transport. We now demonstrate by immunoblotting and immunofluorescence microscopy that platelet MRP4 is absent in two patients with a platelet delta-storage pool deficiency (delta-SPD)-like phenotype with reduced platelet adenine nucleotide (AN) but normal serotonin levels, whereas their other membrane marker proteins of platelet granules were normally expressed and localized. In these patients, MRP4 was present in lymphocytes, and the coding region of their MRP4/ABCC4 gene did not show any mutation that explained the lack of expression. In platelets with classic delta-SPD (low AN and serotonin levels), MRP4 was quantitatively (immunoblot) normal, but, like other delta-granules membrane marker proteins (eg, LAMP2), was mostly displaced from delta-granules to patches at the plasma membrane, suggesting that platelets with classic delta-SPD have an abnormality that impairs the assembly of normal delta-granules. Thus, defective expression of platelet MRP4 is associated with selective defect in AN storage. The genetic basis of the new delta-SPD phenotype remains to be elucidated.

In vitro effects of picotamide on human platelet aggregation, the release reaction and thromboxane B2 production.

We studied the in vitro effects of picotamide (N,N bis 3 picolyl-4-methoxy-isophthalamide) on human platelet aggregation, the release reaction and the production of thromboxane B2 (TxB2) induced by several platelet agonists. The effects of picotamide were compared to those of acetylsalicylic acid (ASA). Picotamide (0.5 mmol/l) inhibited platelet aggregation, the release of ATP and TxB2 production induced by ADP, arachidonic acid (AA), collagen or the prostaglandin endoperoxide (PE) analogue U46619. ASA (0.5 mmol/l) did not affect platelet aggregation and the release of ATP induced by U46619. Picotamide and ASA inhibited the AA-induced platelet TxB2 production both under stirring and non-stirring conditions, whereas the pure thromboxane A2 receptor antagonist BM13177 (0.5 mmol/l) was inhibitory only under stirring conditions. Since under non-stirring conditions platelet aggregation does not occur, picotamide directly inhibits TxB2 production, whereas BM13177 inhibits the potentiation of TxB2 production due to TxA2/PE-dependent platelet aggregation. Malondialdehyde (MDA) production by unstirred platelets stimulated with AA was not significantly inhibited by picotamide. In conclusion, picotamide inhibits the TxA2/PE-dependent platelet responses to agonists by a double mechanism: (i), TxA2/PE antagonism; (ii) inhibition of thromboxane synthase.

Sustained correction of the bleeding time in an afibrinogenaemic patient after infusion of fresh frozen plasma

Summary. We have evaluated the duration of the effect of replacement therapy with two different doses of fibrinogen on the prolonged bleeding time of an afibrinogenaemic patient and the relationship between changes in bleeding time and plasma and platelet fibrinogen concentrations. The infusion of 40 mg/kg fibrinogen, as fresh frozen plasma (FFP). corrected the prolonged bleeding time of the patient from longer that 30 min to 8 min. The bleeding time was still normal 9 d after infusion, at a time when the plasma and platelet fibrinogen levels were low (0.13 g/l and 27 μg/109 platelets; normal ranges 1.6–4.0 and 60–190). Two months later, the infusion of a smaller dose of fibrinogen (4 mg/kg) also corrected the bleeding time, which remained normal until the second day after infusion, despite the fact that plasma and platelet fibrinogen were very low (0.02 g/l and 3.4 μg/109 platelets). The bleeding time returned to the prolonged baseline values only by day 6 post‐infusion, when plasma and platelet fibrinogen levels were 4 × 10–4 g/l and 1.4 μg/109 platelets. Therefore, sustained correction of the prolonged bleeding time may be obtained in afibrinogenaemic patients with a single infusion of fibrinogen at lower doses than usually recommended.