Baseline Platelet Activation Research Articles

In vivo effects of eltrombopag on platelet function in immune thrombocytopenia: no evidence of platelet activation

AbstractThe effects of eltrombopag, a thrombopoietin-receptor agonist, on platelet function in immune thrombocytopenia (ITP) are not fully characterized. This study used whole blood flow cytometry to examine platelet function in 20 patients receiving eltrombopag treatment at days 0, 7, and 28. Platelet surface expression of activated GPIIb/IIIa, P-selectin, and GPIb was measured with and without low and high adenosine diphosphate (ADP) and thrombin receptor activating peptide (TRAP) concentrations. Before eltrombopag treatment with no ex vivo agonist, platelet activation was higher in ITP patients than controls. Platelet GPIb and activated GPIIb/IIIa expression without added agonist was unchanged following eltrombopag treatment, whereas a slight increase in P-selectin was observed. Expression of P-selectin and activated GPIIb/IIIa in response to high-dose ADP was lower during eltrombopag treatment than at baseline. Eltrombopag led to a slight increase in platelet reactivity to TRAP only in responders to eltrombopag but not to levels above those in controls; whole blood experiments demonstrated that this increase was probably because of higher platelet counts rather than higher platelet reactivity. In conclusion, although thrombocytopenic ITP patients have higher baseline platelet activation than controls, eltrombopag did not cause platelet activation or hyper-reactivity, irrespective of whether the platelet count increased.

The influence of aspirin dose and glycemic control on platelet inhibition in patients with type 2 diabetes mellitus

Low-dose aspirin seems to offer no benefit in the primary prevention of cardiovascular disease in type 2 diabetes mellitus (DM2). The anti-platelet effect may be diminished by poor glycemic control or inadequate dosing of aspirin. To study the effects of both glycemic control and increasing aspirin dose on platelet response to aspirin in DM2 patients and matched controls. Platelet effects of increasing doses of aspirin (30, 100 and 300 mg daily) were prospectively assessed in 94 DM2 patients and 25 matched controls by measuring thromboxane levels in urine (11-dhTxB2) and platelet aggregation using VerifyNow(®) and light transmission aggregometry (LTA). DM2 patients were stratified for glycemic control (hemoglobin-A1c [HbA1c] ≤ 53, 53-69, ≥ 69 mmol mol(-1)). At baseline, median 11-dhTxB2 excretion was higher in the poorly controlled patients (77 ng mmol(-1)), and the moderately controlled (84 ng mmol(-1)) compared with the well-controlled patients (64 ng mmol(-1)) and controls (53 ng mmol(-1)), P < 0.01. Next, 30 mg of aspirin reduced 11-dhTxB2 excretion to 31, 29 and 24 ng mmol(-1) in the poorly, moderately and well-controlled patients, respectively, and to 19 ng mmol(-1) in controls, P < 0.001. VerifyNow(®) and LTA were also incompletely suppressed in DM2 patients using 30 mg of aspirin, but 100 mg resulted in similar platelet suppression in all groups, with no additional effect of 300 mg. DM2 patients with inadequate glycemic control (HbA1c > 53 mmol mol(-1)) have higher baseline platelet activity and incomplete suppression of platelet activity with 30 mg of aspirin. However, 100 mg of aspirin leads to optimal inhibition irrespective of glycemic control, and 300 mg does not further improve platelet suppression.

Influence of high‐density lipoprotein and paraoxonase‐1 on platelet reactivity in patients with acute coronary syndromes receiving clopidogrel therapy

The paraoxonase activity of the enzyme paraoxonase-1 (PON-1) associated with high-density lipoprotein (HDL) may significantly influence clopidogrel's antiplatelet and clinical efficacy as a result of its involvement in the clopidogrel biotransformation to the pharmacologically active thiol metabolite. We evaluated the possible relationships of HDL levels as well as PON-1 activities and the Q192R genotype with clopidogrel's antiplatelet efficacy in acute coronary syndrome (ACS) patients. The platelet aggregation, P-selectin expression and platelet/leukocyte conjugates as well as the clopidogrel response variability (evaluated by the VASP phosphorylation test and expressed as platelet reactivity index, PRI) were assessed in 74 ACS patients undergoing percutaneous coronary intervention (PCI) in relation to the PON-1 Q192R genotype and to serum HDL-cholesterol levels, and PON-1 (paraoxonase and arylesterase) activities. Patients were loaded with 600 mg of clopidogrel followed by 75 mg per day. HDL-cholesterol levels and PON-1 activities at baseline (before clopidogrel loading) were not altered at 5- and 30-day post-clopidogrel loading, whereas baseline platelet activation parameters were significantly attenuated. At 5 days, 17 patients were clopidogrel non-responders (PRI: 64.2 ± 11.1%). HDL-cholesterol was inversely associated with platelet activation parameters independently on platelet response variability to clopidogrel whereas a negative association between platelet activation parameters and paraoxonase activity was observed in patients adequately responding to clopidogrel but not in clopidogrel non-responders. Similarly, the platelet activation markers were significantly higher in PON-1 Q192Q genotype carriers compared with those having one or two R alleles only in patients adequately responding to clopidogrel. PON-1 is an important determinant of clopidogrel antiplatelet efficacy only in patients adequately responding to clopidogrel. These findings may be clinically important in ACS patients receiving clopidogrel therapy, especially the first days after the episode.

Mechanistic Study of Hemoglobin Induced Platelet Activation

Abstract 5262Nitric oxide (NO) has been previously shown to inhibit adenosine diphosphate (ADP)- and thrombin- mediated platelet activation. This inhibitory effect of NO is blocked by cell-free oxyhemoglobin (Villagra, Shiva et al. 2007). Cell-free hemoglobin (Hb) also appears to directly activate platelets even in the absence of ADP. However, the precise mechanisms for cell-free Hb-induced platelet activation have not been fully delineated. We hypothesized that oxyhemoglobin can promote platelet activation through one of three potential mechanisms: 1) direct apoprotein interaction with platelet receptors, 2) reactive oxygen species (ROS) production, and/or 3) scavenging of endogenous NO.Platelets were isolated from whole blood obtained from healthy volunteers (n=15) using sodium citrate blood collection tubes. Platelet activation was measured by examining immunofluorescent Pac-1 labeling by flow cytometry using activated glycoprotein IIb/IIIa as a measure of activation. In the presence of 10 uM concentration of NO donor mahmaNONOate, ADP-induced platelet activation was inhibited by 44%. Incubation of 100 uM cell-free oxyhemoglobin eliminated the inhibitory effect of NO. In addition, cell-free oxyhemoglobin (100 uM concentration) induced platelet activation by 17% above unstimulated controls even in the absence of ADP.Methemoglobin was used to test if direct Hb protein interaction with platelets was sufficient for platelet activation, independent of heme redox state. To produce methemoglobin, the ferrous iron in oxyhemoglobin was oxidized to its ferric form, thus methemoglobin is likely to preserve all direct protein interactions. However, unlike oxyhemoglobin, incubation of 100 uM methemoglobin did not induce platelet activation.Next, we tested whether reactive oxygen species production was responsible for hemoglobin-induced platelet activation. In the absence of hemoglobin, superoxide dismutase (SOD) (100 U/ml) and catalase (100 U/ml) reduced baseline platelet activation, verifying a ROS (Pignatelli, Pulcinelli et al. 1998). However, in the presence of oxyhemoglobin, SOD and catalase did not significantly abrogate the effect of Hb on platelet activation, suggesting that the ROS superoxide and hydrogen peroxide are not involved in hemoglobin-induced platelet activation.To determine if scavenging of endogenous NO was responsible for hemoglobin-induced platelet activation, platelets were incubated in the presence or absence of NO scavengers, carboxy-PTIO (5mM) and Fe-DTCS (3 mM), or nitric oxide synthase (NOS) inhibitor, LNMMA (100 uM). Results from these investigations suggest scavenging of endogenous NO was not responsible for hemoglobin-induced platelet activation. Overall our data suggest a complex mechanism for oxyhemoglobin-mediated platelet activation that is likely to involve more than a single contributing pathway.These data aid in elucidating the mechanism of platelet activation by cell-free hemoglobin and may provide novel insights to designing potential treatments to reduce the platelet activation underlying hypercoagulable states associated with a variety of pathological conditions including hemolytic anemias, stroke, and diabetes.This work was supported by NIH grants HL058091 and HL098032 Disclosures:Gladwin:Patents filed related to treating hemolysis.: Patents & Royalties. Kim-Shapiro:Patents filed related to treating hemolysis. Honororia from Sangart Inc.: Honoraria, Patents & Royalties.

The risk of false results in the assessment of platelet function in the absence of antiplatelet medication: Comparision of the PFA-100, multiplate electrical impedance aggregometry and verify now assays

Objectives Evaluation of aspirin (ASA) responsiveness with platelet function tests varies by the choice of blood mixture and functional test and cut off values for defining the the treatment used. Addition to that we also aimed to determine aggregement between three tests and to research whether there is any necessity to measure baseline platelet activity. Methods The study group comprised of 52 patients with multiple risk factors receiving primary prophylaxis of ASA (100 mg/day). For each patient inhibition of platelet aggregation with aspirin was determined using three different whole blood tests: Multiplate electrical impedance aggregometry , Verify Now Aspirin, and collagen-epinephrine closure time PFA-100. Platelet aggregation was assessed with multiplate electrical impedance aggregometry,and was defined as the area under curve (AUC,AUxmin).Maximal 6,4 microM collagen-induced AUC were used to quantify platelet aggregation due to ASA.The ASA response was defined as > 30 % reduction in basal platelet aggregation with multiplate electrical impedance aggregometry.Collagen induced platelet aggregation at the Verify Now Aspirin assay quantitated the ASA-induced platelet inhibition as aspirin reaction units (ARU).According to manifacturer insert ARU > 550 indicates aspirin resistance. ASA platelet function studies were assessed twice at baseline (pre-aspirin), and after 7 day(post-aspirin) were performed. Results After ASA intake none of the patients was found aspirin resistant with PFA-100. (CEPI-CT (129 ± 36 vs 289 ± 18 ). None of the patients was found aspirin resistant with PFA-100. As > 30 % reduction in bazal platelet aggregation with multiplate electrical impedance aggregometry is selected all of the patients have been stratified as responders.(COL TEST 688±230 vs 169 ± 131 AU) None of the patients with Verify Now Aspirin found resistance to ASA(594 ± 62 vs 446 ± 43).Prior to ASA intake 15 of all patients with VN(501 ± 16) and 2 of all patients with multiplate electrical impedance aggregometry (223 ± 40 AUC )aggregation levels below the cut off label before ingestion of ASA.None of the patients was above the cut off label with PFA -100 (129 ± 36). Conclusions Verify Now ASA assay, multiplate electrical impedance aggregometry and PFA-100 seem to be reliable tests in reflecting ASA effect on platelets. Cut off labels for the defining the responsiveness given by manıfacturer may show significant interindividual variabiliy with Verify Now ASA assay and multiplate electrical impedance aggregometry, and these test may show platelet inhibition despite the absence of ASA intake. Consideration of the pretreatment values may eliminate the risk of overestimation in the assessment of platelet inhibtion by ASA.

Platelet Inhibition by Aspirin 81 and 325 mg/day in Men Versus Women Without Clinically Apparent Cardiovascular Disease

Compared with men, women have greater platelet aggregation before and after low-dose aspirin. It is not known whether high-dose aspirin therapy brings residual platelet aggregation in women closer to that in men. Our objective was to compare inhibition of platelet aggregation in women and men after low- and high-dose aspirin. We enrolled healthy subjects (n=106) in a trial of 14 days of aspirin 81 mg/day followed by 14 days of 325 mg/day. Platelet function was measured at baseline and after the 2 aspirin doses. Women had greater baseline platelet activation measurements. After the 2 aspirin doses, men and women had near complete suppression of platelet aggregation to arachidonic acid in whole blood and in platelet-rich plasma (PRP), the direct cyclo-oxygenase-1 pathway affected by aspirin. For indirect pathways, women had significantly greater residual platelet activation to collagen and adenosine diphosphate (ADP) in whole blood after the 2 aspirin doses and in response to collagen and ADP in PRP after aspirin 325 mg/day only. After aspirin 325 mg/day, women continued to have greater residual platelet aggregation compared with men after aspirin 81 mg/day in response to collagen (p=0.016 in whole blood, p=0.037 in PRP), ADP (p<0.001 in whole blood, p=0.012 in PRP), and epinephrine (p=0.03 in PRP). Excretion of urinary thromboxane metabolite (urinary 11-dehydrothromboxane B2) decreased after aspirin to a similar extent in men and women. In conclusion, women continue to have greater residual platelet activity after high-dose aspirin compared with men treated with a lower dose of aspirin.

Baseline platelet activity and response after clopidogrel in 257 diabetics among 822 patients with coronary artery disease

The objective was to describe the indices of platelet aggregation and activation in a large cohort of diabetic patients with coronary artery disease (CAD). Recently, a number of observations have indicated that patients with diabetes mellitus (DM) exhibit persistent platelet activation, and low response after antiplatelet therapy, although no randomized data exist. We sought to define the baseline platelet biomarkers, and the patterns of response to aspirin and clopidogrel therapy in DM versus non-diabetic patients. Secondary post-hoc analyses were made of platelet activity biomarkers in the dataset which consisted of patients with documented CAD (n = 822), including those with DM (n = 257). Patients with DM exhibited higher baseline platelet activity by adenosine diphosphate (ADP)- (p = 0.0002), and collagen-induced (p = 0.03) aggregometry; Ultegra- (p = 0.0001), and PFA-100 (p = 0.02) analyzers; and expression of platelet/endothelial cell adhesion molecule-1 (PECAM-1) (p = 0.01), glycoprotein (GP) IIb/IIIa antigen (p = 0.001), and activity (p = 0.02), vitronectin receptor (p = 0.03), P selectin (p = 0.02), and intact epitope of PAR-1 thrombin receptor (p = 0.02). Antiplatelet response after clopidogrel in diabetics was impaired when compared with non-diabetics. In conclusion, diabetic patients exhibit high pretreatment platelet activity, and do not respond well to the available antiplatelet regimens when compared with similar patients without DM. The clinical implications of these findings are unknown but are potentially important. Considering worsened outcomes in this high-risk population, clinical trials in DM are urgently needed in order to define the optimal degree of platelet inhibition and suitability for more aggressive antiplatelet regimens.

Increased platelet, leukocyte, and coagulation activation in primary myelofibrosis

Platelet and leukocyte activation has been demonstrated in polycythemia vera (PV) and essential thrombocythemia (ET), but such information is limited in primary myelofibrosis (PMF). Platelet, leukocyte, endothelial, and coagulation activation status was assessed in 26 PMF patients and compared with data from 22 age- and sex-matched healthy individuals. Study included flow cytometry assessment of platelet P-selectin expression [at baseline and after adenosine diphosphate (ADP), thrombin and arachidonic acid stimulation], platelet-neutrophil and platelet-monocyte complexes, and CD11b expression in neutrophils and monocytes. Additionally, soluble P-selectin, sCD40L, tissue factor, thrombomodulin, prothrombin fragment 1 + 2 (F1 + 2), and D-dimer were measured by enzyme-linked immunosorbent assays. The above parameters were correlated with the patients' clinical data and presence of the JAK2 V617F mutation. Compared with controls, PMF patients had increased baseline platelet activation, as shown by significantly higher levels of soluble and platelet P-selectin expression, and also higher percentages of platelet-monocyte complexes. Neutrophil and monocyte CD11b expression was significantly higher in patients with the JAK2 mutation than in those with wild-type allele or the controls. Endothelial and coagulation activation, as demonstrated by increased plasma levels of thrombomodulin and F1 + 2, was also found in PMF, with patients with the JAK2 mutation showing significantly higher values of F1 + 2 than those with wild-type allele. In conclusion, PMF patients have platelet, leukocyte, endothelial, and coagulation activation similar to that in PV and ET. CD11b overexpression and F1 + 2 are correlated with the presence of the JAK2 mutation.

Baseline level of platelet-leukocyte aggregates, platelet CD63 expression, and soluble P-selectin concentration in patientswith posttraumatic stress disorder: A pilot study

Platelets may have an important role in the development of cardiovascular disease (CVD) as a result of chronic stress. We conducted a pilot study to evaluate the effect of posttraumatic stress disorder (PTSD) on baseline platelet activation. Platelet-leukocyte aggregates (PLA) and CD63 expression were measured by flow cytometry, and soluble (s)P-selectin concentration was determined in sera of 20 Croatian male combat veterans with PTSD and 20 healthy civilians. Groups were matched in sex, age, body mass index (BMI) and traditional CVD risk factors. Our data showed no differences in measured parameters. Other platelet activation markers should be determined and a larger sample size used in future studies.

Mechanisms of Platelet Activation by Antithymocyte Globulin (ATG).

We have previously shown that polyclonal rabbit ATG from Fresenius (ATG-F) can induce non-overt DIC in patients undergoing hematopoietic stem cell transplantation (HSCT) [Weber et al. 2003]. The compensated state of the coagulopathy suggested that the severe drop in platelet counts (>60%) observed in patients with normal pretreatment levels was not solely due to thrombin generation and platelet consumption. We thus further investigated the mechanisms of ATG-F-induced thrombocytopenia, hypothesizing that ATG-F had direct platelet-stimulating activity. Using an indirect flow cytometric binding assay we found that, compared to control IgG, ATG-F dose-dependently (1–100 μg/ml) bound to the surface of resting and TRAP-activated platelets, showing an up to 50fold increase in MFI at 50–100 μg/ml. In a washed platelet system, in which baseline positivity for CD62P was generally <15%, ATG-F alone had no effect on platelet aggregation. However, when platelets were primed with low concentrations of ADP (1–2 μM) or epinephrine (0.5 μM), ATG-F induced strong and stable aggregation of up to 80–100% in a dose-dependent manner (25–100 μg/ml), whereas control IgG did not. The priming effect of ADP was dependent on both P2Y1 and P2Y12 as evidenced by respective ADP receptor antagonists. Preincubation of platelets with inhibitory CD32 mAb completely abolished ATG-F-induced platelet aggregation, suggesting that clustering of, and signalling through, FcγRIIA was crucial for this platelet response. Similarly, ATG-F-derived F(ab′)2 had no effect on ADP-triggered platelet aggregation. Furthermore, preliminary studies indicated that responsiveness of platelet donors to ATG-F was associated with the FcγRIIA-R/R131 polymorphism, which has previously been shown to confer increased in vitro platelet responsiveness to heparin/PF4 antibodies from patients with HIT. In a 14C-serotonin release assay, in which prolonged incubation at 370C under shaking conditions increases baseline platelet activation and ensures constant platelet-platelet contacts, ATG-F (100 μg/ml), but not control IgG, induced strong FcγRIIA-dependent dense-granule release of up to 80%. In contrast, using diluted platelet-rich plasma under static conditions, ATG-F had only minor effects on platelet activation, increasing CD62P+ platelets from 2±1 to 15±5% (n=5). In a cohort of 16 consecutive HSCT patients receiving ATG-F, two patients were identified with normal pretreatment platelet counts and soluble CD62P (sCD62P) plasma levels of >51 ng/ml (mean+2SD of 23 controls). In these patients, an ATG-F-induced drop in platelet count to 38 and 19% of baseline was associated with a 1.7 and 2.2fold increase in sCD62P, respectively, indicating further platelet activation. In summary, strong binding of ATG-F to resting platelets may accelerate their clearance by the reticulo-endothelial system, thus contributing to the pathophysiology of thrombocytopenia. While ATG-F alone had negligible effects on resting platelets, it significantly enhanced activation of prestimulated platelets. Patients with normal-to-high platelet counts and evidence of in vivo platelet activation may especially be prone to this potentially hazardous side effect.

Cytochrome P450 2C19 loss-of-function polymorphism is a major determinant of clopidogrel responsiveness in healthy subjects

AbstractThe capacity of clopidogrel to inhibit ADP-induced platelet aggregation shows wide intersubject variability. To determine whether frequent functional variants of genes coding for candidate cytochrome P450 (CYP) isoenzymes involved in clopidogrel metabolic activation (CYP2C19*2, CYP2B6*5, CYP1A2*1F, and CYP3A5*3 variants) influence the platelet responsiveness to clopidogrel, we conducted a prospective pharmacogenetic study in 28 healthy white male volunteers treated for 7 days with clopidogrel 75 mg/d. We observed that pharmacodynamic response to clopidogrel was significantly associated with the CYP2C19 genotype. Twenty of the subjects were wild-type CYP2C19 (*1/*1) homozygotes, while the other 8 subjects were heterozygous for the loss-of-function polymorphism CYP2C19*2 (*1/*2). Baseline platelet activity was not influenced by the CYP2C19 genotype. In contrast, platelet aggregation in the presence of 10 μM ADP decreased gradually during treatment with clopidogrel 75 mg once daily in *1/*1 subjects, reaching 48.9% ± 14.9% on day 7 (P < .001 vs baseline), whereas it did not change in *1/*2 subjects (71.8% ± 14.6% on day 7, P = .22 vs baseline, and P < .003 vs *1/*1 subjects). Similar results were found with VASP phosphorylation. The CYP2C19*2 loss-of-function allele is associated with a marked decrease in platelet responsiveness to clopidogrel in young healthy male volunteers and may therefore be an important genetic contributor to clopidogrel resistance in the clinical setting.

P02 Inherent Variation in Donor Platelet Reactivity Contributes to the Quality of Apheresis Platelets

Introduction Provision of platelets is a major commitment for Blood Services. Iterative cycles of improvements in platelet procurement and storage have lead to a better quality of concentrates, but even the latest generation of apheresis machines may produce activated platelets. Less attention has been paid in determining the contribution of an individual donor's inherent platelet reactivity on the activation state of apheresis platelets. We investigated this donor related variable using a panel of donors from the BLOODOMICS project whose platelet responses to activation through the ADP and collagen pathways were known to represent the widest range of the normal responses. For the current study three groups of donors with responses that were characterised as high (n = 16), low (n = 15) or in the mid‐range (n = 16), were recalled and the activation state of their circulating platelets and donated apheresis platelets were measured.Method To assess the activation state of platelets in the circulation, prior to donation samples were taken using a 21G butterfly needle, without a tourniquet. Aliquots of platelets from the apheresis donation were taken immediately at the end of the procedure. The level of platelet‐bound fibrinogen, a sensitive marker for early‐stage activation was measured using a whole‐blood flow cytometric assay.Results Prior to donation resting bound fibrinogen levels in all subjects ranged from 1.6–11.1% (mean 3.6%), and there was a significant association between this index of baseline platelet activation and the donor's responder status (P = 0.009). The percentage of platelets with bound fibrinogen in the apheresis concentrates ranged from 0.6–84.3% (mean 14.7%) and were generally higher than resting levels. Increased apheresis‐induced activation was associated with the responder status of the donor (P = 0.005). In particular, the 4 platelet concentrates with the highest levels of bound fibrinogen (&gt;50%) were all from high responder donors.Conclusion Overall it is not solely the process of donation that affects platelet quality. Platelets that show higher responses to agonists are particularly susceptible to activation during collection by apheresis. We postulate that the responses to agonists are largely genetically controlled, and are attempting to identify the relationship between variation in platelet‐gene sequence and platelet function.

Residual Arachidonic Acid–Induced Platelet Activation via an Adenosine Diphosphate–Dependent but Cyclooxygenase-1– and Cyclooxygenase-2–Independent Pathway

Thrombotic events still occur in aspirin-treated patients with coronary artery disease. To better understand aspirin "resistance," serum thromboxane B2 (TXB2) and flow cytometric measures of arachidonic acid-induced platelet activation (before and after the ex vivo addition of aspirin and indomethacin) were analyzed in 700 consecutive aspirin-treated patients undergoing cardiac catheterization. In 680 of 682 evaluable patients, serum TXB2 concentrations were reduced compared with nonaspirinated healthy donors. Twelve patients had serum TXB2 that was lower than nonaspirinated healthy donors but >10 ng/mL. Arachidonic acid stimulated greater platelet activation in patients with high serum TXB2 (>10 ng/mL) than in patients with low serum TXB2. Addition of ex vivo aspirin reduced arachidonic acid-induced platelet activation to similar levels regardless of serum TXB2 concentrations, which suggests that patients with high residual serum TXB2 concentrations were either noncompliant or underdosed with aspirin. Among the remaining 98% of patients, ex vivo administration of either aspirin or indomethacin failed to prevent platelet activation across all degrees of arachidonic acid-induced platelet activation and aspirin doses. Although the patients were not randomized with respect to clopidogrel treatment, multivariate analysis showed that arachidonic acid-induced platelet activation was less in patients receiving clopidogrel. There is a residual arachidonic acid-induced platelet activation in aspirin-treated patients that (1) is caused by underdosing and/or noncompliance in only approximately 2% of patients and (2) in the remaining patients, occurs via a cyclooxygenase-1 and cyclooxygenase-2 independent pathway, in direct proportion to the degree of baseline platelet activation, and is mediated in part by adenosine diphosphate-induced platelet activation.

IN VIVO EVALUATION OF A BIOMIMETIC ENDOTHELIAL SURFACE FOR EXTRACORPOREAL CIRCULATION

The development of nonthrombogenic surfaces for extracorporeal circulation (ECC) is ongoing. Using nitric oxide (NO)-generating coatings could reduce the need for systemic heparinization during ECC and the complications associated with this therapy. An NO-generating polymer coating using a copper complex to prevent thrombosis was evaluated using a rabbit arteriovenous (AV) model. New Zealand White rabbits were placed on an AV circuit for 4 hours with an intravenous line and arterial line to maintain hemodynamic stability and collect blood samples (arterial blood gases, platelet counts, fibrinogen levels and platelet aggregometry). The custom AV circuit was constructed of 36cm Tygon® tubing with a 14GA angiocatheter (Jelco®) for left carotid artery access and a modified 10 Fr. thoracic catheter (Argyle Kendall) for drainage into the right jugular vein. The lipophilic copper complex generates NO in the presence of S-nitrosothiols and nitrites that occur naturally in blood. The copper complex was incorporated into plasticized polyvinylchloride to coat the entire circuit to generate NO at the blood-biomaterial surface. Two groups were studied: Copper (n=9) and uncoated control (n=5). The copper and control groups had a 48% and 66% reduction in platelet counts, respectively. The copper and control groups maintained 37% and 6% of baseline platelet activity, respectively. The addition of a copper NO catalyst to a polymer coating in contact with blood reduces platelet activation and thrombus formation.

Inhibition of platelet activation by 5-aminosalicylic acid in inflammatory bowel disease.

Platelets play an important role in inflammation and are activated in inflammatory bowel disease. Micro-vascular thrombosis in the gut wall leading to intestinal micro-infarction may be a pathogenic feature of Crohn's disease. 5-Aminosalicylic acid is an effective treatment for patients with inflammatory bowel disease. To assess the effects of 5-aminosalicylic acid on platelet activation, when taken orally and in vitro by patients with inflammatory bowel disease. Spontaneous and thrombin-induced platelet activation were studied using fluorescent antibodies to the activated platelet surface glycoprotein P-selectin and flow cytometry. Baseline platelet activation in inflammatory bowel disease was significantly greater than that in controls (P=0.0003). Independent of diagnosis or disease activity, spontaneous ex-vivo platelet activation was 50% lower in patients with inflammatory bowel disease taking 5-aminosalicylic acid orally than in those not on such treatment (P < 0.05). In vitro, 5-aminosalicylic acid significantly reduced both spontaneous (P < 0. 03 for >/=1 microM 5-aminosalicylic acid) and thrombin-induced platelet activation (P < 0.02 for >/= 1 microM 5-aminosalicylic acid). 5-Aminosalicylic acid given either orally or in vitro inhibits platelet activation. If this effect reflects an in vivo action in the gut, it could contribute to the beneficial actions of 5-aminosalicylic acid in inflammatory bowel disease.

Antecedent Aspirin Therapy Inhibits Baseline Platelet Activity in Patients Presenting with Acute Myocardial Infarction

Aspirin therapy and platelet inhibition reduce the risk for the development of acute myocardial infarction (AMI). However, the effects of aspirin on baseline platelet activity in patients presenting with AMI are not known. We determined the effect of long-term aspirin use on baseline platelet activity in patients presenting with AMI, enrolled in the GUSTO-III Platelet Study. Platelet characteristics were investigated by aggregometry, flow cytometry and enzyme-linked immunosorbent assay in 23 patients before thrombolysis. Sixteen AMI patients were aspirin free, and 7 patients were using aspirin (81–500 mg/daily). The aspirin-treated patients exhibited a mild but consistent reduction of platelet activity which reached significance for 5 µM (p = 0.02), and 10 µM (p = 0.01) adenosine diphosphate induced aggregation. The surface expression of P-selectin (p = 0.02) and PECAM-1 (p = 0.03) and the plasma level of soluble P-selectin (p = 0.02) were also reduced. As previously observed in normal humans and patients with stable coronary artery disease, long-term aspirin therapy is also associated with diminished platelet activation in patients presenting with AMI. Long-term aspirin therapy mildly reduces baseline platelet activity; however, this degree of relative platelet inhibition does not appear to be cardioprotective.

Exaggerated platelet reactivity in major depression.

This study investigated whether depressed patients exhibit exaggerated platelet reactivity. In vivo platelet activation, secretion, and dose-response aggregation were measured in 12 depressed patients and eight normal comparison subjects after overnight bed rest and following orthostatic challenge. The depressed patients exhibited increased platelet activation at baseline, demonstrated by increased binding of monoclonal antibody (moAb) annexin V protein reacting with prothrombinase complex binding sites. Following orthostatic challenge, the depressed patients exhibited increases in binding of moAbs PAC1 and anti-LIBS1 against activated glycoprotein IIb/IIIa and GE12 against P-selectin expressed upon secretion. The normal comparison subjects exhibited increases in platelet activation only with GE12. Depressed patients exhibit enhanced baseline platelet activation and responsiveness in comparison with normal subjects. Heightened susceptibility to platelet activation may be a mechanism by which depression is a significant risk factor for ischemic heart and cerebrovascular disease and/or mortality after myocardial infarction.

Platelet changes after a saturated fat meal and their prevention by Dazmegrel, a thromboxane synthetase inhibitor.

A saturated fat meal was given to 10 normal males both before and after pretreatment with UK38,485, a thromboxane synthetase inhibitor (Dazmegrel, Pfizer Central Research, Sandwich, Kent, United Kingdom). Following the fat meal at 2 and 4 hr there was laboratory evidence of platelet activation. Dazmegrel lowered baseline platelet activity and lessened platelet activation following the fat meal. This drug should be investigated further as a potential antithrombotic agent.