Prevention of P2 Receptor-Dependent Thrombocyte Activation by Pore-Forming Bacterial Toxins Improves Outcome in A Murine Model of Urosepsis.

Urosepsis is a potentially life-threatening, systemic reaction to uropathogenic bacteria entering the bloodstream of the host. One of the hallmarks of sepsis is early thrombocyte activation with a following fall in circulating thrombocytes as a result of intravascular aggregation and sequestering of thrombocytes in the major organs. Development of a thrombocytopenic state is associated with a poorer outcome of sepsis. Uropathogenic Escherichia coli frequently produce the pore-forming, virulence factor α-haemolysin (HlyA), of which the biological effects are mediated by ATP release and subsequent activation of P2 receptors. Thus, we speculated that inhibition of thrombocyte P2Y1 and P2Y12 receptors might ameliorate the septic response to HlyA-producing E. coli. The study combined in vitro measurements of toxin-induced thrombocyte activation assessed as increased membrane abundance of P-selectin, fibronectin and CD63 and data from in vivo murine model of sepsis-induced by HlyA-producing E. coli under infusion of P2Y1 and P2Y12 antagonists. Our data show that the P2Y1 receptor antagonist almost abolishes thrombocyte activation by pore-forming bacterial toxins. Inhibition of P2Y1, by constant infusion of MRS2500, markedly increased the survival in mice with induced sepsis. Moreover, MRS2500 partially prevented the sepsis-induced depletion of circulating thrombocytes and dampened the sepsis-associated increase in proinflammatory cytokines. In contrast, P2Y12 receptor inhibition had only a marginal effect in vivo and in vitro. Taken together, inhibition of the P2Y1 receptor gives a subtle dampening of the thrombocyte activation and the cytokine response to bacteraemia, which may explain the improved survival observed by P2Y1 receptor antagonists.

Evaluation of platelet function in thrombocytopenia.

Whole blood aggregometry is a functional assay for determination of platelet function. Until now, whole blood aggregometry has not been considered feasible at low platelet counts. Hence, the objectives of the present study were to explore platelet function in thrombocytopenia using a novel index of impedance aggregometry adjusted for platelet count and evaluate the association to platelet function assessed by flow cytometry. Hirudin anticoagulated blood was collected from 20 healthy volunteers, 20 patients with primary immune thrombocytopenia (ITP), and 17 hematological cancer patients. Platelet function was analyzed by impedance aggregometry and by flow cytometry. Collagen, adenosine diphosphate, thrombin receptor agonist peptide-6, and ristocetin were used as agonists for both analyses. Thrombocytopenia in healthy whole blood was induced in vitro employing a recently published method. Platelet aggregation of thrombocytopenic patients was evaluated relative to the aggregation of healthy volunteers at the same platelet count. In flow cytometry, platelet function was described as expression of the platelet surface glycoproteins: bound fibrinogen, CD63, and P-selectin. Similar platelet counts were obtained in the patient groups (p = 0.69) (range: 13-129 × 109/l). Aggregation adjusted for platelet count was significantly increased in ITP patients compared to healthy platelets across all agonists. The platelet aggregation was high in the 95% prediction interval, with 18 ITP patients above the prediction interval in at least two agonists. In contrast, the platelet aggregation was low in the prediction interval in cancer patients, and three cancer patients with platelet aggregation below the prediction interval in at least one agonist. ITP patients displayed increased expression of bound fibrinogen and CD63 following activation, compared with particularly cancer patients, but also compared with healthy platelets. This study demonstrated the feasibility of a novel approach to perform platelet function analyses in thrombocytopenia using impedance aggregometry adjusted for platelet count.

Flow cytometric analysis of platelet cyclooxygenase-1 and -2 and surface glycoproteins in patients with immune thrombocytopenia and healthy individuals.

Immature platelets may contain more platelet enzymes such as cyclooxygenase (COX)-1 and COX-2 than mature platelets. Patients with immune thrombocytopenia (ITP) have a higher fraction of immature platelets and can therefore be utilized as a biological model for investigating COX-1 and COX-2 platelet expression. The aims were to develop flow cytometric assays for platelet COX-1 and COX-2 and to investigate the COX-1 and COX-2 platelet expression, platelet turnover, and platelet glycoproteins in ITP patients (n = 10) compared with healthy individuals (n = 30). Platelet count and platelet turnover parameters (mean platelet volume (MPV), immature platelet fraction (IPF), and immature platelet count (IPC)) were measured by flow cytometry (Sysmex XE-5000). Platelet COX-1, COX-2, and the glycoproteins (GP)IIb, IX, Ib, Ia, and IIIa were all analyzed by flow cytometry (Navios) and expressed as median fluorescence intensity. COX analyses were performed in both whole blood and platelet rich plasma (PRP), whereas platelet glycoproteins were analyzed in whole blood only. ITP patients had significantly lower platelet count (55 × 109/L) than healthy individuals (240 × 109/L, p < 0.01), but a higher MPV (p = 0.03) and IPF (p < 0.01). IPC was similar for the two groups (p = 0.74). PRP had significantly lower MPV (p < 0.01) and significantly higher platelet count and IPC (both p-values <0.03) when compared with whole blood. IPF was similar for PRP and whole blood (p = 0.18). COX-1 expression was 10 times higher and COX-2 expression was 50% higher in PRP than in whole blood (pCOX-1 < 0.01, pCOX-2 < 0.01). Platelet COX-1 expression was higher in ITP patients than healthy individuals using whole blood (pCOX-1 < 0.01) and PRP, though this was nonsignificant in PRP (pCOX-1 = 0.17). In ITP patients, positive correlations were found between platelet turnover and COX-1 expression (all p-values <0.01, rho = 0.80-0.94), whereas healthy individuals showed significant though weaker correlations between platelet turnover and COX-1 and COX-2 expressions (all p-values <0.03, rho = 0.44-0.71). GPIIb, IX, and Ib expression was increased in ITP patients compared with healthy individuals (all p-values < 0.03). GPIIb, IX, Ib, and IIIa showed positive correlations with platelet turnover in ITP patients (all p-values <0.02, rho = 0.71-0.94), but weak and nonsignificant correlations in healthy individuals (all p-values >0.14, rho = 0.11-0.28). In conclusion, ITP patients expressed higher COX-1 and platelet glycoprotein levels than healthy individuals. COX-1 and platelet glycoproteins demonstrated positive correlations with platelet turnover in ITP patients. In healthy individuals, COX-1 and COX-2 expression correlated positively with platelet turnover. PRP was more sensitive compared with whole blood as regards determination of COX. Therefore, PRP is the recommended matrix for investigating COX-1 and COX-2 in platelets.

Thrombocytopenia model with minimal manipulation of blood cells allowing whole blood assessment of platelet function.

In vitro models of thrombocytopenia are useful research tools. Previously published models have shortcomings altering properties of platelets and other blood components. The aim of the present study was to develop a whole blood method to induce thrombocytopenia with minimal manipulation, and to describe platelet function in induced thrombocytopenia in individuals with healthy platelets. Hirudin anticoagulated blood was obtained from 20 healthy volunteers. One part of the blood was gently centrifuged at 130g for 15 minutes. The platelet-rich plasma was replaced with phosphate-buffered saline to establish thrombocytopenia. Various levels of thrombocytopenia were achieved by combining different volumes of baseline whole blood and thrombocytopenic blood. Platelet counts were measured by flow cytometry (Navios, Beckman Coulter) and routine haematological analyser (Sysmex XE-5000). Platelet function was analysed by impedance aggregometry (Multiplate® Analyzer, Roche) and by flow cytometry (Navios, Beckman Coulter) using collagen, adenosine diphosphate, thrombin receptor activating peptide-6 and ristocetin as agonists. Median baseline platelet count was 227×10(9)/l. The in vitro model yielded median platelet counts at 51×10(9)/l (range 26-93×10(9)/l). We observed minor, yet significant, changes in platelet size and maturity from baseline to modelled thrombocytopenia. In the thrombocytopenic samples, significant and positive linear associations were found between platelet count and platelet aggregation across all agonists (all p-values<0.001). Platelet function assessed by flow cytometry showed minimal alterations in the thrombocytopenic samples. A new whole blood-based model of thrombocytopenia was established and validated. This new model serves as a useful future tool, particularly to explore platelet function in patients with thrombocytopenia.

Investigation of platelet function and platelet disorders using flow cytometry.

Patients with thrombocytopenia or platelet disorders are at risk of severe bleeding. We report the development and validation of flow cytometry assays to diagnose platelet disorders and to assess platelet function independently of platelet count. The assays were developed to measure glycoprotein levels (panel 1) and platelet function (panel 2) in sodium citrated blood. Twenty healthy volunteers and five patients diagnosed with different platelet disorders were included. Glycoprotein expression levels of the receptors Ia, Ib, IIb, IIIa and IX were measured and normalised with forward scatter (FS) as a measurement of platelet size. Platelet function was assessed by CD63, P-selectin and bound fibrinogen in response to arachidonic acid, adenosine diphosphate (ADP), collagen-related peptide, ristocetin and thrombin receptor-activation peptide-6. All patients except one with suspected δ-granule defect showed aberrant levels of glycoproteins in panel 1. Glanzmann's thrombasthenia and genetically verified Bernard-Soulier syndrome could be diagnosed using panel 1. All patients showed reduced platelet function according to at least one agonist. Using panel 2 it was possible to diagnose Bernard-Soulier syndrome, δ-granule defect and GPVI disorder. By combining the two assays, we were able to diagnose different platelet disorders and investigate platelet function independent of platelet count.

Rapid evaluation of platelet function using the Multiplate® Analyzer.

Rapid evaluation of platelet function may be advantageous in the setting of surgical and interventional procedures to tailor treatment of ongoing bleeding. We investigated if platelet function testing performed with the Multiplate® Analyzer (Roche Diagnostics, Mannheim, Germany) only 5 minutes after blood sampling yields reliable test results compared to analyses performed 30 minutes after sampling as currently recommended. We included 48 patients with type II diabetes and stable coronary artery disease treated with aspirin 75 mg daily and 50 healthy individuals not taking any medications. Platelet aggregometry by the Multiplate® Analyzer was performed 5 and 30 minutes after blood sampling using arachidonic acid (1.0 mM), collagen (3.2 µg/ml) and adenosine diphosphate (ADP; 6.5 µM) as agonists. Compliance with aspirin was verified by serum thromboxane B2 measurements. Aggregation levels assessed 5 minutes after blood sampling correlated strongly with those assessed after 30 minutes irrespective of the agonist used (r-values 0.75-0.89, p values <0.0001). Aggregation levels were 4-8% lower and displayed a larger standard deviation when measured 5 minutes after sampling, compared to 30 minutes after sampling. Weak, but significant correlations were observed between platelet aggregation and platelet count (r-values = 0.28-0.39; p values <0.01). The currently recommended 30-minute standing time can be omitted, when platelet aggregation is measured using the Multiplate® Analyzer. Platelet aggregation measured 5 minutes after blood sampling correlates strongly with aggregation measured 30 minutes after sampling, but yields slightly lower aggregation levels. The Multiplate® Analyzer enables rapid on-site evaluation of platelet function.

Low-dose acetylsalicylic acid therapy monitored with ultra high performance liquid chromatography.

OBJECTIVES: Assessment of compliance in patients on low-dose acetylsalicylic acid (ASA) therapy is limited to interview, pill counting, witnessed ASA intake, and measurement of thromboxane B2 levels. We validated a new, sensitive assay for analyzing blood levels of ASA and the metabolite salicylic acid (SA). DESIGN AND METHODS: Blood samples were withdrawn from 10 healthy volunteers and 50 patients with stable coronary artery disease, before and after intake of 75 mg ASA. Plasma was mixed with acetonitrile, and 2-methylbenzoic acid was added as internal standard. After filtration, ASA and SA were quantified with ultra high performance liquid chromatography (UHPLC). Separation was accomplished with an isocratic flow of acetonitrile in phosphate buffer pH2.5, and a Zorbax RRHD C18 analytical column. Detection was achieved with wavelength photodiode array detection set at 237 nm. RESULTS: The ASA- and SA-assay showed linearity (r(2)>0.999) within the range of 0.2-200 µg/mL, and with detection limits of 170 ng/mL and 53 ng/mL. The intra- and inter-day imprecision for ASA and SA were 2.2-5.9%, 3.4-11.7% and 1.8-8.0%, 3.8-9.4%, respectively. Recovery was between 89 and 103% for both assays. More than 60 coadministered drugs were investigated for interference, and only one drug interfered with the ASA-assay and none with the SA-assay. ASA was measurable 1h after intake of 75 mg ASA, and SA was measurable 1 and 6h after ASA intake. CONCLUSION: We developed a fast and reliable UHPLC assay with a high sensitivity and selectivity, suitable for monitoring of compliance in patients treated with low-dose ASA.

Reference intervals for platelet aggregation assessed by multiple electrode platelet aggregometry.

INTRODUCTION: Analyses of platelet aggregation in hirudin whole blood using Multiplate® was validated. Reference intervals for the most commonly used agonists were established, and the association between platelet aggregation, age, gender and haematological values was analysed. MATERIAL AND METHODS: We included 121 healthy individuals to establish reference intervals and six healthy individuals for evaluation of the day-to-day variation. Platelet aggregation was evaluated on hirudin whole blood employing Multiplate® induced by arachidonic acid, ADP, collagen and ristocetin (RISTOlow and RISTOhigh). Measurements of haematological values were performed employing Sysmex K-4500. RESULTS: We found no association between platelet aggregation and age (p>0.57 for all agonists, except RISTOlow: p=0.05). Platelet aggregation was significantly higher in women compared to men for all agonists (p<0.0003), except RISTOlow (p=0.05). A reference interval is presented as 95% confidence interval suitable for any age and both sex. Day-to-day variation was <11% for all agonists except for RISTOlow. No association was found between platelet aggregation and haematocrit or red blood cell count after adjusting for age and gender except for RISTOhigh. A positive significant association was found between platelet count and platelet aggregation (p<0.04). Finally, a significant positive association was found between platelet aggregation and white blood cell count for all agonists (p<0.05) except RISTOlow and RISTOhigh (p>0.05). CONCLUSION: Reference intervals for platelet aggregation in healthy individuals (age: 17 to 66 years) were established in hirudin whole blood measured by Multiplate® employing the most commonly used agonists.