Decreased platelet activation predicts hepatic decompensation and mortality in patients with cirrhosis.

BACKGROUND AND AIMS: Patients with cirrhosis show alterations in primary hemostasis, yet prognostic implications of changes in platelet activation remain controversial, and assay validity is often limited by thrombocytopenia. We aimed to study the prognostic role of platelet activation in cirrhosis, focusing on bleeding/thromboembolic events, decompensation, and mortality. APPROACH AND RESULTS: We prospectively included 107 patients with cirrhosis undergoing a same-day hepatic venous pressure gradient (HVPG) and platelet activation measurement. Platelet activation was assessed using flow cytometry after protease-activated receptor (PAR)-1, PAR-4, or epinephrine stimulation. Over a follow-up of 25.3 (IQR: 15.7-31.2) months, first/further decompensation occurred in 29 patients and 17 died. More pronounced platelet activation was associated with an improved prognosis, even after adjusting for systemic inflammation, HVPG, and disease severity. Specifically, higher PAR-4-inducible platelet activation was independently linked to a lower decompensation risk [adjusted HR per 100 MFI (median fluorescence intensity): 0.95 (95% CI: 0.90-0.99); p =0.036] and higher PAR-1-inducible platelet activation was independently linked to longer survival [adjusted HR per 100 MFI: 0.93 (95% CI: 0.87-0.99); p =0.040]. Thromboembolic events occurred in eight patients (75% nontumoral portal vein thrombosis [PVT]). Higher epinephrine-inducible platelet activation was associated with an increased risk of thrombosis [HR per 10 MFI: 1.07 (95% CI: 1.02-1.12); p =0.007] and PVT [HR per 10 MFI: 1.08 (95% CI: 1.02-1.14); p =0.004]. In contrast, of the 11 major bleedings that occurred, 9 were portal hypertension related, and HVPG thus emerged as the primary risk factor. CONCLUSIONS: Preserved PAR-1- and PAR-4-inducible platelet activation was linked to a lower risk of decompensation and death. In contrast, higher epinephrine-inducible platelet activation was a risk factor for thromboembolism and PVT.

Microvascular Thrombosis as a Critical Factor in Severe COVID-19.

Platelet-endothelial interactions have a critical role in microcirculatory function, which maintains tissue homeostasis. The subtle equilibrium between platelets and the vessel wall is disturbed by the coronavirus disease 2019 (COVID-19), which affects all three components of Virchow's triad (endothelial injury, stasis and a hypercoagulable state). Endotheliitis, vasculitis, glycocalyx degradation, alterations in blood flow and viscosity, neutrophil extracellular trap formation and microparticle shedding are only few pathomechanisms contributing to endothelial damage and microthrombosis resulting in capillary plugging and tissue ischemia. In the following opinion paper, we discuss major pathological processes leading to microvascular endothelial activation and thrombosis formation as a possible major adverse factor driving the deterioration of patient disease course in severe COVID-19.

Protease-activated receptor-mediated platelet aggregation in patients with type 2 diabetes on potent P2Y12 inhibitors.

BACKGROUND: Antiplatelet therapy is a cornerstone in the secondary prevention of ischemic events following percutaneous coronary intervention (PCI). The new P2Y12 receptor inhibitors prasugrel and ticagrelor have been shown to improve patients' outcomes. Whether or not these drugs have equal efficacy in individuals with or without diabetes is disputed. Furthermore, platelets can be activated by thrombin, which is, at least in part, independent of P2Y12 -mediated platelet activation. Protease-activated receptor (PAR)-1 and -4 are thrombin receptors on human platelets. We sought to compare the in vitro efficacy of prasugrel (n = 121) and ticagrelor (n = 99) to inhibit PAR-mediated platelet aggregation in individuals with type 2 diabetes (prasugrel n = 26, ticagrelor n = 29). MATERIALS AND METHODS: We compared P2Y12 -, PAR-1- and PAR-4-mediated platelet aggregation as assessed by multiple electrode platelet aggregometry between prasugrel- and ticagrelor-treated patients without and with type 2 diabetes who underwent acute PCI. RESULTS: Overall, there were no differences of P2Y12 -, PAR-1- and PAR-4-mediated platelet aggregation between prasugrel- and ticagrelor-treated patients. However, both drugs inhibited P2Y12 -mediated platelet aggregation stronger, and thereby to a similar extent in patients with type 2 diabetes than in those without diabetes. There was no correlation between either P2Y12 -, or PAR-1- or PAR-4-mediated platelet aggregation and levels of HbA1c or the body mass index (BMI). However, we observed patients with high residual platelet reactivity in response to PAR-1 and PAR-4 stimulation in all cohorts. CONCLUSION: Prasugrel and ticagrelor inhibit P2Y12 - and PAR-mediated platelet aggregation in individuals with diabetes to a similar extent, irrespective of HbA1c levels and BMI.

Peripheral versus central venous blood sampling does not influence the assessment of platelet activation in cirrhosis.

Cirrhotic patients have an increased risk of bleeding and thromboembolic events, with platelets being involved as key players in both situations. The impact of peripheral versus central blood sampling on platelet activation remains unclear. In 33 cirrhotic patients, we thus analyzed platelet function in peripheral (P) and central (C) blood samples. Platelet surface expression of P-selectin, activated glycoprotein (GP) IIb/IIIa, and leukocyte-platelet aggregate formation were measured by flow cytometry in response to different agonists: thrombin receptor-activating peptide-6, adenosine diphosphate, collagen-related peptide (CrP), epinephrine, AYPGKF, Pam3CSK4, and lipopolysaccharide. Unstimulated platelet surface expression of P-selectin (p = .850) and activated GPIIb/IIIa (p = .625) were similar in peripheral and central blood samples. Stimulation with various agonists yielded similar results of platelet surface expression of P-selectin and activated GPIIb/IIIa in peripheral and central samples, except for CrP-inducible expression of activated GPIIb/IIIa (median fluorescence intensity, MFI in P: 7.61 [0.00-24.66] vs. C: 4.12 [0.00-19.04], p < .001). The formation of leukocyte-platelet aggregate was similar in central and peripheral blood samples, both unstimulated and after stimulation with all above-mentioned agonists. In conclusion, peripheral vs. central venous blood sampling does not influence the assessment of platelet activation by flow cytometry in cirrhosis.Abbreviations: ACLD: advanced chronic liver disease; ADP: adenosine diphosphate; ALD: alcoholic liver disease; AYPGKF: PAR-4 agonist AYPGKF; CrP: collagen related protein; EPI: epinephrine; FACS: fluorescence-activated cell sorting; GP: glycoprotein; HVPG: hepatic venous pressure gradient; IQR: interquartile range; LPS: lipopolysaccharide; LSM: liver stiffness measurement; MFI: median fluorescence intensity; NAFLD: nonalcoholic fatty liver disease; PAM: lipopeptide Pam3CSK4; PAR: protease-activated receptor; PBS: phosphate-buffered saline; PH: portal hypertension; TIPS: transjugular intrahepatic portosystemic stent shunt; TLR: toll-like receptor; TRAP-6: thrombin receptor-activator peptide-6; vWF: von Willebrand factor.

Platelet activation and aggregation in different centrifugal-flow left ventricular assist devices.

Left-ventricular assist devices (LVADs) improve outcomes in end-stage heart failure patients. Two centrifugal-flow LVAD systems are currently approved, HeartMate 3 (HM3) and Medtronic/Heartware HVAD (HVAD). Clinical findings suggest differences in thrombogenicity between both systems. We compared markers of platelet activation and aggregation between HM3 and HVAD. We prospectively included 59 LVAD patients (40 HM3, 19 HVAD). Platelet P-selectin expression, activated glycoprotein (GP) IIb/IIIa and monocyte-platelet aggregates (MPA) were assessed by flow-cytometry. Platelet aggregation was measured by light-transmission aggregometry (LTA) and multiple-electrode aggregometry (MEA). Von-Willebrand factor (VWF) antigen (VWF:Ag), VWF activity (VWF:Ac), and VWF multimer pattern analysis were determined. Soluble P-selectin (sP-selectin) was measured with an enzyme-linked immunoassay. P-selectin, GPIIb/IIIa and MPA levels in vivo and in response to arachidonic acid, adenosine diphosphate, and thrombin receptor activating peptide were similar between HM3 and HVAD (all p > .05). Likewise, agonist-inducible platelet aggregation by LTA and MEA did not differ between HM3 and HVAD (all p > .05). VWF:Ag levels and FVIII:C were similar between both systems (both p > .05), but patients with HVAD had significantly lower VWF:Ac (p = .011) and reduced large VWF multimers (p = .013). Finally, sP-selectin levels were similar in patients with HVAD and HM3 (p = .845). In conclusion, on-treatment platelet activation and aggregation are similar in HM3 and HVAD patients. Potential clinical implications of observed differences in VWF profiles between both LVAD systems need to be addressed in future clinical trials.

Angiotensin-Converting Enzyme Inhibitors and Angiotensin Receptor Blockers in Acute Coronary Syndrome: Implications for Platelet Reactivity?

BACKGROUND: In patients with acute coronary syndrome (ACS), angiotensin-converting enzyme (ACE) inhibitors are preferred over angiotensin receptor blockers (ARBs). However, in a recent pilot study, treatment with ACE inhibitors was associated with increased platelet reactivity compared to ARBs. Therefore, we sought to investigate the impact of renin-angiotensin-aldosterone system (RAAS) blockade with ACE inhibitors and ARBs on platelet aggregation in patients with ACS undergoing percutaneous coronary intervention. METHODS: On-treatment residual platelet reactivity in response to arachidonic acid (AA), adenosine diphosphate (ADP), SFLLRN, AYPGKF, and collagen was assessed by multiple electrode aggregometry (MEA) in 197 ACS patients on dual antiplatelet therapy (DAPT) with aspirin and either prasugrel or ticagrelor. RESULTS: One hundred sixty-five (83.7%) patients were treated with ACE inhibitors, 32 (16.3%) with ARBs. On-treatment residual AA- and ADP-inducible platelet reactivity was significantly higher in patients with ACE inhibitors (both p < 0.05). Likewise, SFLLRN was significantly higher in patients with ACE inhibitors (p = 0.036) and there was a trend for higher AYPGKF- and collagen-inducible platelet reactivity (p = 0.053 and p = 0.082). The incidence of high on-treatment residual platelet reactivity AA was significantly higher in patients with ACE inhibitors (52 [31.5%] vs. 3 [9.4%] patients; p = 0.019). CONCLUSION: ACE inhibitors are associated with increased on-treatment residual platelet reactivity in ACS patients with potent DAPT. Further clinical trials are needed to elucidate the role of RAAS blockade with ACE inhibitors and ARBs in ACS patients treated according to current standards.

Decreased Platelet Inhibition by Thienopyridines in Hyperuricemia.

PURPOSE: Hyperuricemia carries an increased risk of atherothrombotic events in acute coronary syndrome (ACS) patients undergoing percutaneous coronary intervention (PCI). This may at least in part be due to inadequate P2Y12 inhibition. The aim of this study was to prospectively investigate the potential association between hyperuricemia and decreased platelet inhibition by P2Y12 antagonists. METHODS: Levels of uric acid as well as on-treatment residual platelet reactivity in response to adenosine diphosphate (ADP) were assessed in 301 clopidogrel-treated patients undergoing elective angioplasty and stenting, and in 206 prasugrel- (n = 118) or ticagrelor-treated (n = 88) ACS patients following acute PCI. Cut-off values for high on-treatment residual ADP-inducible platelet reactivity (HRPR) were based on previous studies showing an association of test results with clinical outcomes. RESULTS: Hyperuricemia was significantly associated with increased on-treatment residual ADP-inducible platelet reactivity in clopidogrel- and prasugrel-treated patients in univariate analyses and after adjustment for differences in patient characteristics by multivariate regression analyses. In contrast, ticagrelor-treated patients without and with hyperuricemia showed similar levels of on-treatment residual platelet reactivity to ADP. HRPR occurred more frequently in clopidogrel- and prasugrel-treated patients with hyperuricemia than in those with normal uric acid levels. In contrast, hyperuricemic patients receiving ticagrelor did not have a higher risk of HRPR compared with those with normal uric acid levels. CONCLUSION: Hyperuricemia is associated with decreased platelet inhibition by thienopyridines but a normal response to ticagrelor. It remains to be established if lowering uric acid increases the antiplatelet effects of clopidogrel and prasugrel in hyperuricemic patients with HRPR.

Ticagrelor Inhibits Toll-Like and Protease-Activated Receptor Mediated Platelet Activation in Acute Coronary Syndromes.

PURPOSE: Since ticagrelor inhibits the cellular uptake of adenosine, thereby increasing extracellular adenosine concentration and biological activity, we hypothesized that ticagrelor has adenosine-dependent antiplatelet properties. In the current study, we compared the effects of ticagrelor and prasugrel on platelet activation in acute coronary syndrome (ACS). METHODS: Platelet surface expression of P-selectin and activated glycoprotein (GP) IIb/IIIa in response to adenosine diphosphate (ADP), the toll-like receptor (TLR)-1/2 agonist Pam3CSK4, the TLR-4 agonist lipopolysaccharide (LPS), the protease-activated receptor (PAR)-1 agonist SFLLRN, and the PAR-4 agonist AYPGKF were measured by flow cytometry in blood from 80 ticagrelor- and 80 prasugrel-treated ACS patients on day 3 after percutaneous coronary intervention. Residual platelet aggregation to arachidonic acid (AA) and ADP were assessed by multiple electrode aggregometry and light transmission aggregometry. RESULTS: ADP-induced platelet activation and aggregation, and AA-induced platelet aggregation were similar in patients on ticagrelor and prasugrel, respectively (all p ≥ 0.3). Further, LPS-induced platelet surface expression of P-selectin and activated GPIIb/IIIa did not differ significantly between ticagrelor- and prasugrel-treated patients (both p > 0.4). In contrast, Pam3CSK4-induced platelet surface expression of P-selectin and activated GPIIb/IIIa were significantly lower in ticagrelor-treated patients (both p ≤ 0.005). Moreover, SFLLRN-induced platelet surface expression of P-selectin and activated GPIIb/IIIa were significantly less pronounced in patients on ticagrelor therapy compared to prasugrel-treated patients (both p < 0.03). Finally, PAR-4 mediated platelet activation as assessed by platelet surface expression of activated GPIIb/IIIa following stimulation with AYPGKF was significantly lower in patients receiving ticagrelor (p = 0.02). CONCLUSION: Ticagrelor inhibits TLR-1/2 and PAR mediated platelet activation in ACS patients more strongly than prasugrel.

Podoplanin expression in primary brain tumors induces platelet aggregation and increases risk of venous thromboembolism.

Venous thromboembolism (VTE) is common in patients with brain tumors, and underlying mechanisms are unclear. We hypothesized that podoplanin, a sialomucin-like glycoprotein, increases the risk of VTE in primary brain tumors via its ability to induce platelet aggregation. Immunohistochemical staining against podoplanin and intratumoral platelet aggregates was performed in brain tumor specimens of 213 patients (mostly high-grade gliomas [89%]) included in the Vienna Cancer and Thrombosis Study, a prospective observational cohort study of patients with newly diagnosed cancer or progressive disease aimed at identifying patients at risk of VTE. Platelet aggregation in response to primary human glioblastoma cells was investigated in vitro. During 2-year follow-up, 29 (13.6%) patients developed VTE. One-hundred fifty-one tumor specimens stained positive for podoplanin (33 high expression, 47 medium expression, 71 low expression). Patients with podoplanin-positive tumors had lower peripheral blood platelet counts (P < .001) and higher D-dimer levels (P < .001). Podoplanin staining intensity was associated with increasing levels of intravascular platelet aggregates in tumor specimens (P < .001). High podoplanin expression was associated with an increased risk of VTE (hazard ratio for high vs no podoplanin expression: 5.71; 95% confidence interval, 1.52-21.26; P =010), independent of age, sex, and tumor type. Podoplanin-positive primary glioblastoma cells induced aggregation of human platelets in vitro, which could be abrogated by an antipodoplanin antibody. In conclusion, high podoplanin expression in primary brain tumors induces platelet aggregation, correlates with hypercoagulability, and is associated with increased risk of VTE. Our data indicate novel insights into the pathogenesis of VTE in primary brain tumors.

Oral antiplatelet agents in cardiovascular disease.

Antiplatelet agents significantly reduce mortality and morbidity in ischemic heart disease, cerebrovascular disease and peripheral artery disease (PAD), and are therefore part of guideline-driven daily medical treatment in these patients. Due to its beneficial effects in the secondary prevention of atherothrombotic events, aspirin remains the most frequently prescribed antiplatelet agent in cardiovascular disease. In patients with acute coronary syndromes (ACS) and in those undergoing angioplasty with stent implantation dual antiplatelet therapy with aspirin and an adenosine diphosphate (ADP) receptor antagonist is indicated. The development of the newer ADP P2Y12 inhibitors prasugrel and ticagrelor has further improved prognosis in ACS patients compared to clopidogrel. Moreover, vorapaxar allows the inhibition of platelet activation by thrombin via protease-activated receptor-1 and has been approved for the use in patients with PAD and in those with a history of myocardial infarction. This review article summarizes the current evidence on oral antiplatelet agents in cardiovascular disease. Keywords: Aspirin, clopidogrel, prasugrel, ticagrelor, vorapaxar, cardiovascular disease.

Plasma Levels of snoRNAs are Associated with Platelet Activation in Patients with Peripheral Artery Disease.

In addition to supervised walking therapy, antithrombotic therapy and the management of risk factors, the treatment of peripheral artery disease (PAD) is limited to endovascular and surgical interventions, i.e., angioplasty with stent implantation and bypass surgery, respectively. Both are associated with a high restenosis rate. Furthermore, patients with PAD often suffer atherothrombotic events like myocardial infarction, transient ischemic attacks or stroke. Small ribonucleic acids (RNAs) have proven reliable biomarkers because of their remarkable stability. Small nucleolar RNAs (snoRNAs) guide modifications to small nuclear RNAs and ribosomal RNAs, enabling protein synthesis. In the current study, we measured four snoRNAs in 104 consecutive PAD patients who underwent elective infrainguinal angioplasty with stent implantation. We selected snoRNAs that showed significant overexpression in the plasma of end-stage PAD patients in a previous study. All four snoRNAs are transcribed from the 14q32 locus, which is strongly linked to human cardiovascular disease, including PAD and restenosis. We showed that the four selected 14q32 snoRNAs were abundantly expressed in the plasma of PAD patients. The plasma levels of these snoRNAs were not directly associated with target vessel restenosis, however, levels of SNORD113.2 and SNORD114.1 were strongly linked to platelet activation, which is an important determinant of long-term outcome, in PAD, and in cardiovascular disease in general.

Impaired glucose metabolism is associated with increased thrombin generation potential in patients undergoing angioplasty and stenting.

BACKGROUND: As a strong platelet agonist on the one hand and key molecule in plasmatic coagulation on the other hand, thrombin connects primary and secondary hemostasis. Thrombin generation potential reflects the individual capacity to generate thrombin, and has been associated with the occurrence of thromboembolic events. In the current study, we sought to identify predictors of thrombin generation potential in patients undergoing angioplasty and stenting for atherosclerotic cardiovascular disease. METHODS: Peak thrombin generation potential and area under the curve (AUC) of thrombin generation potential were determined with a commercially available assay in 315 patients on dual antiplatelet therapy 1 day after percutaneous intervention, and in 100 healthy individuals without cardiovascular disease. RESULTS: Median (interquartile range) peak thrombin generation potential and AUC of thrombin generation potential in the study cohort (n = 315) were significantly higher than in healthy individuals (n = 100) without cardiovascular disease (peak thrombin generation potential: 445.4 nM [354.5-551.8 nM] vs. 174.5 nM [141.2-261.2 nM]; AUC of thrombin generation potential: 5262.7 nM thrombin [4806.6-5756.9 nM thrombin] vs. 3405.2 nM thrombin [3043.6-3747.3 nM thrombin]; both p < 0.001). In patients undergoing angioplasty and stenting, hemoglobin A1c (HbA1c) was the only variable that was independently associated with both, peak thrombin generation potential and AUC of thrombin generation potential (both p ≤ 0.007). In contrast, platelet count and high-sensitivity C-reactive protein were only associated with peak thrombin generation potential, and body mass index and serum creatinine were only associated with AUC of thrombin generation potential after adjustment for covariates by multivariate linear regression analyses (all p < 0.05). Patients with HbA1c ≥ 6% had significantly higher peak thrombin generation potential and AUC of thrombin generation potential than patients with HbA1c < 6% (peak thrombin generation potential: 476.9 nM [385.8-577.9 nM] vs. 423.9 nM [335.8-529.5 nM], p = 0.002; AUC of thrombin generation potential: 5371.8 nM thrombin [4903 - 5899 nM thrombin] vs. 5172.5 nM thrombin [4731.8-5664.7 nM thrombin], p = 0.01). HbA1c ≥ 6% remained independently associated with both parameters of thrombin generation potential after multivariate linear regression analyses (both p ≤ 0.02). CONCLUSIONS: Impaired glucose metabolism is associated with increased thrombin generation potential in patients undergoing angioplasty and stenting for cardiovascular disease.

Decreased platelet inhibition by P2Y12 receptor blockers in anaemia.

BACKGROUND: Anaemic patients undergoing angioplasty and stenting are at an increased risk of ischaemic events, which may be caused by an inadequate response to antiplatelet therapy with adenosine diphosphate (ADP) P2Y12 inhibitors. In the current study, we investigated the associations between anaemia and on-treatment platelet reactivity in clopidogrel-treated (group 1, n = 306) and prasugrel-/ticagrelor-treated (group 2, n = 109) patients undergoing elective and acute angioplasty with stent implantation, respectively. MATERIALS AND METHODS: Monocyte-platelet aggregate (MPA) formation was determined by flow cytometry in both groups. On-treatment residual platelet reactivity in response to ADP was assessed by light transmission aggregometry (LTA) in both groups, and by the VerifyNow P2Y12 assay and the Impact-R in group 1. P-selectin expression was measured by flow cytometry in group 2. RESULTS: In both groups, anaemia was associated with significantly higher MPA formation in response to ADP (both P ≤ .02). Moreover, by LTA maximal aggregation in response to ADP was significantly higher in patients with anaemia in both groups (both P < .05), and anaemic patients in group 1 had a significantly higher on-treatment platelet reactivity by the VerifyNow P2Y12 assay and the Impact-R than those without anaemia (both P < .001). In group 2, significantly higher platelet surface expression of P-selectin was seen in anaemia after stimulation with ADP (P = .02). CONCLUSION: Anaemia is associated with decreased platelet inhibition by ADP P2Y12 receptor antagonists after elective and acute percutaneous interventions with stent implantation. However, due to inconsistencies between different platelet function tests additional data are needed to clarify the role of anaemia for platelet inhibition.

Granulocyte antibodies in male blood donors: can they trigger transfusion-related acute lung injury?

BACKGROUND: White blood cell-associated antibodies can lead to transfusion-related acute lung injury (TRALI). Female donors with a history of pregnancies have been identified as a main cause for these antibodies. Male or female donors without a history of pregnancy are considered as safe donors. STUDY DESIGN AND METHODS: Following the identification of two TRALI cases associated with blood products from male donors, we investigated the frequency of granulocyte-specific and human leukocyte antigen (HLA) antibodies in the entire blood donor population using a high throughput automated flow-cytometry-based granulocyte immunofluorescence test (Flow-GIFT). We investigated sera from 14,343 whole blood donors (female, n = 6974, 48.7%; male, n = 7369, 51.3%) using automated Flow-GIFT. Of the female blood donors, 60.4% had a history of pregnancy. Positive sera were retested by the standard granulocyte immunofluorescence test and granulocyte agglutination test. For the detection of HLA Class I and II immunoglobulin G antibodies, we used a commercial screening enzyme-linked immunosorbent assay. RESULTS: We detected in 924 (21.9%) of the 4212 females with a history of pregnancy antibodies against granulocyte antigens (n = 62, 1.5%), HLA Class I and/or II antigens (n = 864, 20.5%). Notably, in 3.5% (n = 96) of 2762 females without a history of pregnancy and in 2.1% (n = 154) of 7369 males antibodies against granulocyte antigens (n = 13, 0.47% and n = 45, 0.6%), HLA Class I and/or II (n = 83, 3% and n = 109, 1.4%, respectively), were also detected. CONCLUSION: Human neutrophil antigen antibodies are rare in male and females without a history of pregnancy compared to females with a history of pregnancy, but their relevance is not negligible.

Platelets Toll-like receptor-4 in Crohns disease.

BACKGROUND: Platelets are activated in Crohn's disease (CD) and interplay with leukocytes. Engagement of Toll-like receptor-4 (TLR-4), which is expressed in human platelets, may be involved in crosstalks between platelets and leukocytes leading to their mutual activation for host defense. MATERIALS AND METHODS: Human neutrophil peptides (HNPs), lipoprotein binding peptides, and sCD14 were determined by enzyme-linked immunosorbent assays in 42 patients with active CD, in 43 patients with CD in remission, and in 30 healthy individuals. Neutrophil-platelet aggregates and binding of the TLR-4 monoclonal antibody to platelets were determined by flow cytometry. RESULTS: Levels of HNPs were higher in patients with CD than in controls (P = 0.0003 vs. active CD and P = 0.01 vs. CD in remission). Likewise, neutrophils with adhering platelets were higher in patients with active CD than in controls (P = 0.004). Binding of the TLR-4 antibody in patients with active CD was similar to that in controls, while patients in remission had significantly higher binding capacities (P = 0.59 and P = 0.003). Incubation of plasma from patients with active disease or patients in remission with platelets from healthy controls confirmed lower binding of the TLR-4 antibody in the presence of plasma from active diseased patients compared to controls (P = 0.039), possibly due to high levels of lipopolysaccharides, as suggested by high levels of sCD14 and lipoprotein binding protein. CONCLUSION: Our study indicates involvement of platelet TLR-4 in enhancing the secretion of antimicrobial peptides from neutrophils. While platelet aggregation can be due to a variety of mechanisms in inflammatory disease, the mutual activation of platelets and neutrophils may augment host defense.

Fibrinolysis in patients with a mild-to-moderate bleeding tendency of unknown cause.

In more than 50% of patients with a mild-to-moderate bleeding tendency, no underlying cause can be identified (bleeding of unknown cause, BUC). Data on parameters of fibrinolysis in BUC are scarce in the literature and reveal discrepant results. It was the aim of this study to investigate increased fibrinolysis as a possible mechanism of BUC. We included 270 patients (227 females, median age 44 years, 25-75th percentile 32-58) with BUC and 98 healthy controls (65 females, median age 47 years, 25-75thpercentile 39-55). Tissue plasminogen activator (tPA-) antigen and activity, plasminogen activator inhibitor type-1 (PAI-1), tPA-PAI-1 complexes, thrombin activatable fibrinolysis inhibitor (TAFI), α2-antiplasmin, and D-dimer were determined. While PAI-1 deficiency was equally frequent in patients with BUC and controls (91/270, 34%, and 33/98, 34%, p = 0.996), tPA activity levels were more often above the detection limit in patients than in controls (103/213, 48%, and 23/98, 23%, p < 0.0001). We found lower levels of tPA-PAI-1 complexes (6.86 (3.99-10.00) and 9.11 (7.17-13.12), p < 0.001) and higher activity of TAFI (18.61 (15.80-22.58) and 17.03 (14.02-20.02), p < 0.001) and α2-antiplasmin (102 (94-109) and 98 (90-106], p = 0.003) in patients compared to controls. Detectable tPA activity (OR 3.02, 95%CI 1.75-5.23, p < 0.0001), higher levels of TAFI (OR 2.57, 95%CI 1.48-4.46, p = 0.0008) and α2-antiplasmin (OR 1.03, 95%CI 1.01-1.05, p = 0.011), and lower levels of tPA-PAI-1 complexes (OR 0.90, 95%CI 0.86-0.95, p < 0.0001) were independently associated with BUC in sex-adjusted logistic regression analyses. We conclude that the fibrinolytic system can play an etiological role for bleeding in patients with BUC.

Underlying mechanism and specific prevention of hemolysis-induced platelet activation.

Thromboembolic complications significantly impair the outcome of hemolytic disorders. We hypothesized that red cell adenosine diphosphate (ADP) release results in significant platelet activation in hemolysis and that this prothrombotic state can be prevented by inhibition of the ADP P2Y12 receptor. In the current study, we therefore sought to investigate the mechanism and inhibition of hemolysis-induced platelet activation. The expression of activated integrin αIIbß3 was determined by flow cytometry, and platelet aggregation was assessed by multiple electrode platelet aggregometry. We demonstrate platelet activation and increased platelet aggregation by adding hemolytic blood (lysates) to whole blood, similarly to that achieved by the platelet agonist ADP. Enhanced platelet activation and reactivity in the presence of hemolytic blood were significantly abolished by apyrase, which catalyzes ADP degradation, and inhibited by blockade of the platelet ADP P2Y12 receptor with cangrelor. Platelets from patients treated with the ADP P2Y12 receptor antagonist clopidogrel showed a reduced response to lysates compared to platelets from healthy controls without antiplatelet treatment. Further, in vitro blood group ABO incompatibility induced hemolysis and led to increased platelet activation. Finally, "spontaneous" platelet aggregation seen in patients with cold agglutinin disease was completely abolished by cangrelor. In conclusion, hemolysis is associated with increased platelet activation and aggregation due to red cell derived ADP, which can be prevented by ADP receptor blockade.

Antibodies against complement-regulatory proteins on platelets in immune thrombocytopenia.

In immune thrombocytopenia (ITP), antibodies reacting with platelet membrane glycoproteins (GP) mediate premature platelet cleavage, resulting in thrombocytopenia and therefore a risk of bleeding. These antibodies may induce complement activation, thus mediating complement-induced platelet destruction. In this study, we investigated the possibility of an additional complement-related pathogenic mechanism, where antibodies against the complement-regulatory factors CD55 and CD59 may directly interfere with normal complement function. CD55 downregulates both the classic and the alternative activation pathways, while CD59 blocks the formation of the membrane attack complex; both proteins are present on platelets and may therefore be targets of autoantibodies. Using the simultaneous analysis of specific platelet antibodies (SASPA) assay, we found that in some cases of immune-mediated thrombocytopenia, anti-CD55 and -CD59 antibodies are detectable in patients' sera and/or on their autologous platelets in combination with antibodies against platelet-specific GP. Although antibodies against CD55 and CD59 seem to be a rare phenomenon, this finding may have clinical relevance due to the availability of highly effective therapeutics targeting the complement system.

ß-blockers are associated with decreased leucocyte-platelet aggregate formation and lower residual platelet reactivity to adenosine diphosphate after angioplasty and stenting.

BACKGROUND: The beneficial effects of ß-blockers on the long-term prognosis of patients with cardiovascular disease may in part be attributable to decreased platelet activation. In this prospective cohort study, we sought to investigate the impact of concomitant ß-blocker therapy on sensitive markers of platelet activation and aggregation. MATERIALS AND METHODS: Monocyte-platelet (MPA) and neutrophil-platelet aggregate (NPA) formation in vivo and in response to the platelet agonist adenosine diphosphate (ADP) were determined by flow cytometry in 258 patients undergoing angioplasty and stenting. On-treatment residual platelet reactivity to ADP was assessed by multiple electrode aggregometry (MEA). RESULTS: One hundred seventy-five patients of the study population (67·8%) received ß-blockers. Treatment with ß-blockers was associated with significantly lower MPA and NPA formation in vivo and in response to ADP compared to patients without ß-blockers (all P ≤ 0·01). The inverse associations of MPA and NPA formation with ß-blocker therapy remained statistically significant after adjustment for differences in patient characteristics by multivariate linear regression analyses (all P < 0·05). Moreover, high levels of MPA in response to ADP as well as high levels of NPAin vivo and in response to ADP were significantly less frequent in patients with ß-blocker treatment (all P < 0·05). Finally, on-treatment residual platelet reactivity to ADP by MEA was significantly lower in patients receiving ß-blockers (P = 0·005). CONCLUSION: ß-Blockers are associated with decreased leucocyte-platelet aggregate formation and lower on-treatment residual platelet reactivity to ADP in patients with dual antiplatelet therapy following angioplasty and stenting.

Serum Cholinesterase Levels Are Associated With 2-Year Ischemic Outcomes After Angioplasty and Stenting for Peripheral Artery Disease.

PURPOSE: To prospectively investigate the associations of serum cholinesterase (CHE) levels with ischemic outcomes after angioplasty and stenting for lower limb peripheral artery disease (PAD). METHODS: A prospective cohort study enrolled 108 patients with Rutherford category 2-3 ischemia who had successful primary unilateral angioplasty and self-expanding bare metal stent implantation for superficial femoral artery (SFA) stenosis. The primary endpoint was a composite of nonfatal myocardial infarction, nonfatal stroke or transient ischemic attack, cardiovascular death, or >80% target lesion restenosis within 2 years after peripheral angioplasty. Target lesion restenosis (restenosis endpoint) and the composite of the aforementioned atherothrombotic events (atherothrombotic endpoint) within 2-year follow-up were defined as secondary endpoints. RESULTS: CHE levels were not available in 4 patients due to technical problems and 4 patients were lost to follow-up. The remaining 100 patients (median age 65 years; 62 men) met the minimum sample size requirement for statistical analysis. Median CHE levels were significantly lower in patients who subsequently experienced the primary endpoint compared with patients without ischemic events [7.1 (IQR 6.3-8.1) vs 8 (IQR 7-9.3) kU/L, p=0.007]. A CHE level <8.3 kU/L was identified as the best cutoff value to predict the primary endpoint, providing an 82.1% sensitivity and 44.3% specificity. The primary endpoint occurred significantly more often in patients with low CHE <8.3 kU/L than in patients with higher CHE levels (32 vs 7 patients, p=0.01). In multivariable Cox regression analysis, low CHE was associated with a 2.6-fold increased risk (95% CI 1.1 to 5.9, p=0.03) of the primary endpoint. Moreover, patients who suffered the secondary restenosis endpoint had significantly lower median CHE levels than patients without restenosis [7.1 (IQR 6.3-8.2) vs 7.9 (IQR 7-8.9) kU/L, p=0.02], and restenosis occurred more frequently in patients with low CHE compared with those with higher CHE levels (27 vs 7 patients, p=0.04). CONCLUSION: Low CHE is associated with an increased risk of long-term adverse ischemic events following SFA angioplasty with stent implantation for PAD.