Acute coronary syndrome remodels the antiplatelet aggregation properties of HDL particle subclasses

Essentials

• HDL subclasses were studied in acute coronary syndrome (ACS).
• HDL2 from ACS patients have better antiplatelet potency than HDL from non ACS subjects.
• ACS remodels the antiplatelet properties of HDL subclasses.
• Oxidized polyunsaturated fatty acids content of HDL is modified by ACS.

Summary

Background

Although HDLs have antithrombotic effects by reducing platelet activation, the relationship between HDL levels and the risk of acute coronary syndrome (ACS) is unclear, as HDL particles are heterogeneous in composition and biological properties.

Objective

To characterize the effects of HDL2 and HDL3 subclasses from ACS patients and non‐coronary artery disease (CAD) subjects on platelet activation.

Methods

We measured platelet aggregation and ex vivo thrombus formation, analyzed signaling pathways by flow cytometry, and performed a targeted lipidomics analysis on HDL subclasses.

Results

Analysis of human platelet aggregation in suspension, adhesion on von Willebrand factor and thrombus formation on collagen under arterial shear demonstrated that HDL2 from ACS patients had higher antiplatelet potency than HDL3 from ACS patients and HDL from non‐CAD subjects. HDL binding to scavenger receptor class B type I was essential for this effect. A lipidomics analysis revealed that HDL2 from ACS patients had more oxidized polyunsaturated fatty acids (PUFAs). An inverse correlation between the concentrations of 9‐hydroxyoctadecadienoic acid (9‐HODE), 13‐hydroxyoctadecadienoic acid (13‐HODE), the eicosapentaenoic acid metabolite 18‐hydroxyeicosapentaenoic acid (18‐HEPE) and hydroxyeicosatetraenoic acid isomers in HDL2 and platelet aggregation was observed. This relationship was further demonstrated by the direct inhibitory effects of 18‐HEPE, 9‐HODE, 13‐HODE, 17‐hydroxydocosahexaenoic acid and 14‐hydroxydocosahexaenoic acid on collagen‐related peptide‐induced platelet aggregation, indicating that oxidized PUFAs contribute to the antithrombotic effect of ACS HDL2.

Conclusions

Our data shed new light on the antiplatelet effects of HDL subclasses, and suggest physiological adaptation through the modulation of HDL properties in ACS patients that may limit their platelet‐dependent thrombotic risk.

     

Preoperative platelet transfusions to reverse antiplatelet therapy for urgent non‐cardiac surgery: an observational cohort study

Essentials

• An increasing number of patients requiring surgery receive antiplatelet therapy (APT).
• We analyzed 181 patients receiving presurgery platelet transfusions to reverse APT.
• No coronary thrombosis occurred after platelet transfusion.
• This justifies a prospective trial to test preoperative platelet transfusions to reverse APT.

Summary

Background

Patients receiving antiplatelet therapy (APT) have an increased risk of perioperative bleeding and cardiac adverse events (CAE). Preoperative platelet transfusions may reduce the bleeding risk but may also increase the risk of CAE, particularly coronary thrombosis in patients after recent stent implantation.

Objectives

To analyze the incidence of perioperative CAE and bleeding in patients undergoing non‐cardiac surgery using a standardized management of transfusing two platelet concentrates preoperatively and restart of APT within 24–72 h after surgery.

Methods

A cohort of consecutive patients on APT treated with two platelet concentrates before non‐cardiac surgery between January 2012 and December 2014 was retrospectively identified. Patients were stratified by the risk of major adverse cardiac and cerebrovascular events (MACCE). The primary objective was the incidence of CAE (myocardial infarction, acute heart failure and cardiac troponine T increase). Secondary objectives were incidences of other thromboembolic events, bleedings, transfusions and mortality.

Results

Among 181 patients, 88 received aspirin, 21 clopidogrel and 72 dual APT. MACCE risk was high in 63, moderate in 103 and low in 15 patients; 67 had cardiac stents. Ten patients (5.5%; 95% CI, 3.0–9.9%) developed a CAE (three myocardial infarctions, four cardiac failures and three troponin T increases). None was caused by coronary thrombosis. Surgery‐related bleeding occurred in 22 patients (12.2%; 95% CI, 8.2–17.7%), making 12 re‐interventions necessary (6.6%; 95% CI, 3.8–11.2%).

Conclusion

Preoperative platelet transfusions and early restart of APT allowed urgent surgery and did not cause coronary thromboses, but non‐thrombotic CAEs and re‐bleeding occurred. Randomized trials are warranted to test platelet transfusion against other management strategies.

     

McMaster RARE‐Bestpractices clinical practice guideline on diagnosis and management of the catastrophic antiphospholipid syndrome

Summary

Background

The McMaster RARE‐Bestpractices project group selected the catastrophic antiphospholipid syndrome (CAPS) for a pilot exercise in guideline development for a rare disease.

Objectives

The objectives of this exercise were to provide a proof of principle that guidelines can be developed for rare diseases and assist in clinical decision making for CAPS.

Patients/Methods

The GIN‐McMaster Guideline Development checklist and GRADE methodology were followed throughout the guideline process. The CAPS guideline was coordinated by a steering committee, and the guideline panel was formed with representation from all relevant stakeholder groups. Systematic reviews were performed for the key questions. To supplement the published evidence, we piloted novel methods, including use of an expert‐based evidence elicitation process and ad hoc analysis of registry data.

Results

This paper describes the CAPS guideline recommendations, including evidence appraisal and discussion of special circumstances and implementation barriers identified by the panel. Many of these recommendations are conditional, because of subgroup considerations in this heterogeneous disease, as well as variability in patient values and preferences.

Conclusions

The CAPS clinical practice guideline initiative met the objective of the successful development of a clinical practice guideline in a rare disease using GRADE methodology. We expect that clinicians caring for patients with suspected CAPS will find the guideline useful in assisting with diagnosis and management of this rare disease.

     

Platelet rescue by macrophage depletion in obese ADAMTS‐13‐deficient mice at risk of thrombotic thrombocytopenic purpura

Essentials

• Obesity is a potential risk factor for development of thrombotic thrombocytopenic purpura (TTP).
• Obese ADAMTS‐13‐deficient mice were triggered with von Willebrand factor (VWF).
• Depletion of hepatic and splenic macrophages protects against thrombocytopenia in this model.
• VWF enhances phagocytosis of platelets by macrophages, dose‐dependently.

Summary

Background

Thrombotic thrombocytopenic purpura (TTP) is caused by the absence of ADAMTS‐13 activity. Thrombocytopenia is presumably related to the formation of microthrombi rich in von Willebrand factor (VWF) and platelets. Obesity may be a risk factor for TTP; it is associated with abundance of macrophages that may phagocytose platelets.

Objectives

To evaluate the role of obesity and ADAMTS‐13 deficiency in TTP, and to establish whether macrophages contribute to thrombocytopenia.

Methods

Lean or obese ADAMTS‐13‐deficient (Adamts‐13^?/?) and wild‐type (WT) mice were injected with 250 U kg^?1 of recombinant human VWF (rVWF), and TTP characteristics were evaluated 24 h later. In separate experiments, macrophages were depleted in the liver and spleen of lean and obese WT or Adamts‐13^?/? mice by injection of clodronate‐liposomes, 48 h before injection of rVWF.

Results

Obese Adamts‐13^?/? mice had a lower platelet count than their lean counterparts, suggesting that they might be more susceptible to TTP development. Lean Adamts‐13^?/? mice triggered with a threshold dose of rVWF did not develop TTP, whereas typical TTP symptoms developed in obese Adamts‐13^?/? mice, including severe thrombocytopenia and higher lactate dehydrogenase (LDH) levels. Removal of hepatic and splenic macrophages by clodronate injection in obese Adamts‐13^?/? mice before treatment with rVWF preserved the platelet counts measured 24 h after the trigger. In vitro experiments with cultured macrophages confirmed a VWF dose‐dependent increase of platelet phagocytosis.

Conclusions

Obese Adamts‐13^?/? mice are more susceptible to the induction of TTP‐related thrombocytopenia than lean mice. Phagocytosis of platelets by macrophages contributes to thrombocytopenia after rVWF injection in this model.

     

Evaluation of a microfluidic flow assay to screen for von Willebrand disease and low von Willebrand factor levels

Essentials

• von Willebrand factor (VWF) function is shear stress dependent.
• Platelet accumulation in a microfluidic assay correlates with VWF levels.
• The microfluidic assay discriminates type 1 von Willebrand disease from healthy controls.
• The microfluidic flow assay detects responses to therapeutic intervention (DDAVP).

Summary

Background

von Willebrand disease (VWD) is a mucocutaneous bleeding disorder with a reported prevalence of 1 in 10 000. von Willebrand factor (VWF) function and platelet adhesion are regulated by hemodynamic forces that are not integrated into most current clinical assays.

Objective

We evaluated whether a custom microfluidic flow assay (MFA) can screen for deficiencies in VWF in patients presenting with mucocutaneous bleeding.

Methods

Whole blood from individuals with mucocutaneous bleeding was assayed in a custom MFA.

Results

Thirty‐two patients with type 1 VWD (10/32) or reported mucocutaneous bleeding were enrolled. The platelet adhesion velocity (r = 0.5978 for 750 s^?1 and 0.6895 for 1500 s^?1) and the maximum platelet surface area coverage (r = 0.5719 for 750 s^?1 and 0.6633 for 1500 s^?1) in the MFA correlated with VWF levels. Furthermore, the platelet adhesion velocity at 750 s^?1 (type 1 VWD, mean 0.0009761, 95% confidence interval [CI] 0.0003404–0.001612; control, mean 0.003587, 95% CI 0.002455–0.004719) and at 1500 s^?1 (type 1 VWD, mean 0.0003585, 95% CI 0.00003914–0.0006778; control, mean 0.003132, 95% CI 0.001565–0.004699) differentiated type 1 VWD from controls. Maximum platelet surface area coverage at 750 s^?1 (type 1 VWD, mean 0.1831, 95% CI 0.03816–0.3281; control, mean 0.6755, 95% CI 0.471–0.88) and at 1500 s^?1 (type 1 VWD, mean 0.07873, 95% CI 0.01689–0.1406; control, mean 0.6432, 95% CI 0.3607–0.9257) also differentiated type 1 VWD from controls. We also observed an improvement in platelet accumulation after 1‐desamino‐8‐d ‐arginine vasopressin (DDAVP) treatment at 1500 s^?1 (pre‐DDAVP, mean 0.4784, 95% CI 0.1777–0.7791; post‐DDAVP, mean 0.8444, 95% CI 0.7162–0.9726).

Conclusions

These data suggest that this approach can be used as a screening tool for VWD.

     

Risk factors for bleeding,including platelet count threshold,in newly diagnosed immune thrombocytopenia adults

Essentials

• Risk factors of bleeding in adult immune thrombocytopenia are not known.
• This multicenter study assessed risk factors of bleeding at immune thrombocytopenia onset.
• Platelet count thresholds associated with bleeding were < 20 × 10⁹ L^?1 and < 10 × 10⁹ L^?1.
• Exposure to anticoagulants was a major risk factor of severe bleeding.

Summary

Background

The aim of this cross‐sectional study was to assess risk factors for bleeding in immune thrombocytopenia (ITP) adults, including the determination of platelet count thresholds.

Methods

We selected all newly diagnosed ITP adults included in the Cytopénies Auto‐immunes Registre Midi‐PyrénéEN (CARMEN) register and at the French referral center for autoimmune cytopenias. The frequencies of any bleeding, mucosal bleeding and severe bleeding (gastrointestinal, intracranial, or macroscopic hematuria) at ITP onset were assessed. Platelet count thresholds were assessed by the use of receiver operating characteristic curves. All potential risk factors were included in logistic regression models.

Results

Among the 302 patients, the frequencies of any, mucosal and severe bleeding were 57.9%, 30.1%, and 6.6%, respectively. The best discriminant threshold of platelet count for any bleeding was 20 × 10⁹ L^?1. In multivariate analysis, factors associated with any bleeding were platelet count (< 10 × 10⁹ L^?1 versus ≥ 20 × 10⁹ L^?1, odds ratio [OR] 48.2, 95% confidence interval [CI] 20.0–116.3; between 10 × 10⁹ L^?1 and 19 × 10⁹ L^?1 versus ≥ 20 × 10⁹ L^?1, OR 5.2, 95% CI 2.3–11.6), female sex (OR 2.6, 95% CI 1.3–5.0), and exposure to non‐steroidal anti‐inflammatory drugs (NSAIDs) (OR 4.8, 95% CI 1.1–20.7). A low platelet count was also the main risk factor for mucosal bleeding. Exposure to anticoagulant drugs was associated with severe bleeding (OR 4.3, 95% CI 1.3–14.1).

Conclusions

Platelet counts of < 20 × 10⁹ L^?1 and < 10 × 10⁹ L^?1 were thresholds for major increased risks of any and mucosal bleeding. Platelet count, female sex and exposure to NSAIDs should be considered for assessment of the risk of any bleeding. Exposure to anticoagulant drugs was a major risk factor for severe bleeding.

     

Small molecule targeting the Rac1‐NOX2 interaction prevents collagen‐related peptide and thrombin‐induced reactive oxygen species generation and platelet activation

Essentials

• Reactive oxygen species (ROS) generation by NOX2 plays a critical role in platelet activation.
• Rac1 regulation of NOX2 is important for ROS generation.
• Small molecule inhibitor of the Rac1‐p67^phox interaction prevents platelet activation.
• Pharmacologic targeting of Rac1‐NOX2 axis can be a viable approach for antithrombotic therapy.

Summary

Background

Platelets from patients with X‐linked chronic granulomatous disease or mice deficient in nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidase isoform NOX2 exhibit diminished reactive oxygen species (ROS) generation and platelet activation. Binding of Rac1 GTPase to p67^phox plays a critical role in NOX2 activation by facilitating the assembly of the NOX2 enzyme complex.

Objective

We tested the hypothesis that Phox‐I, a rationally designed small molecule inhibitor of Rac–p67^phox interaction, may serve as an antithrombosis agent by suppressing ROS production and platelet activation.

Results

Collagen‐related peptide (CRP) induced ROS generation in a time‐dependent manner. Platelets from Rac1^?/? mice or human platelets treated with NSC23766, a specific Rac inhibitor, produced significantly less ROS in response to CRP. Treatment of platelets with Phox‐I inhibited diverse CRP‐induced responses, including: (i) ROS generation; (ii) release of P‐selectin; (iii) secretion of ATP; (iv) platelet aggregation; and (v) phosphorylation of Akt. Similarly, incubation of platelets with Phox‐I inhibited thrombin‐induced: (i) secretion of ATP; (ii) platelet aggregation; (iii) rise in cytosolic calcium; and (iv) phosphorylation of Akt. In mouse models, intraperitoneal administration of Phox‐I inhibited: (i) collagen‐induced platelet aggregation without affecting the tail bleeding time and (ii) in vivo platelet adhesion/accumulation at the laser injury sites on the saphenous vein without affecting the time for complete cessation of blood loss.

Conclusions

Small molecule targeting of the Rac1–p67^phox interaction may present an antithrombosis regimen by preventing GPVI‐ and non‐GPVI‐mediated NOX2 activation, ROS generation and platelet function without affecting the bleeding time.

     

Flavin monooxygenase 3, the host hepatic enzyme in the metaorganismal trimethylamine N‐oxide‐generating pathway,modulates platelet responsiveness and thrombosis risk

Essentials

• Microbe‐dependent production of trimethylamine N‐oxide (TMAO) contributes to thrombosis risk.
• The impact of host flavin monooxygenase 3 (FMO3) modulation on platelet function is unknown.
• Genetic manipulation of FMO3 in mice alters systemic TMAO levels and thrombosis potential.
• Genetic manipulation of FMO3 is associated with alteration of gut microbial community structure.

Summary

Background

Gut microbes play a critical role in the production of trimethylamine N‐oxide (TMAO), an atherogenic metabolite that impacts platelet responsiveness and thrombosis potential. Involving both microbe and host enzymatic machinery, TMAO generation utilizes a metaorganismal pathway, beginning with ingestion of trimethylamine (TMA)‐containing dietary nutrients such as choline, phosphatidylcholine and carnitine, which are abundant in a Western diet. Gut microbial TMA lyases use these nutrients as substrates to produce TMA, which upon delivery to the liver via the portal circulation, is converted into TMAO by host hepatic flavin monooxygenases (FMOs). Gut microbial production of TMA is rate limiting in the metaorganismal TMAO pathway because hepatic FMO activity is typically in excess.

Objectives

FMO3 is the major FMO responsible for host generation of TMAO; however, a role for FMO3 in altering platelet responsiveness and thrombosis potential in vivo has not yet been explored.

Methods

The impact of FMO3 suppression (antisense oligonucleotide‐targeting) and overexpression (as transgene) on plasma TMAO levels, platelet responsiveness and thrombosis potential was examined using a murine FeCl₃‐induced carotid artery injury model. Cecal microbial composition was examined using 16S analyses.

Results

Modulation of FMO3 directly impacts systemic TMAO levels, platelet responsiveness and rate of thrombus formation in vivo. Microbial composition analyses reveal taxa whose proportions are associated with both plasma TMAO levels and in vivo thrombosis potential.

Conclusions

The present studies demonstrate that host hepatic FMO3, the terminal step in the metaorganismal TMAO pathway, participates in diet‐dependent and gut microbiota‐dependent changes in both platelet responsiveness and thrombosis potential in vivo .

     

Genes associated with venous thromboembolism in colorectal cancer patients

Essentials

• The underlying pathophysiological mechanisms behind cancer‐associated thrombosis are unknown.
• We compared expression profiles in tumor cells from patients with and without thrombosis.
• Tumors from patients with thrombosis showed significant differential gene expression profiles.
• Patients with thrombosis had a proinflammatory status and increased fibrin levels in the tumor.

Summary

Background

Venous thromboembolism (VTE) is a frequent complication in patients with cancer, and is associated with significant morbidity and mortality. However, the mechanisms behind cancer‐associated thrombosis are still incompletely understood.

Objectives

To identify novel genes that are associated with VTE in patients with colorectal cancer (CRC).

Methods

Twelve CRC patients with VTE were age‐matched and sex‐matched to 12 CRC patients without VTE. Tumor cells were isolated from surgical samples with laser capture microdissection approaches, and mRNA profiles were measured with next‐generation RNA sequencing.

Results

This approach led to the identification of new genes and pathways that might contribute to VTE in CRC patients. Application of ingenuity pathway analysis indicated significant links with inflammation, the methionine degradation pathway, and increased platelet function, which are all key processes in thrombus formation. Tumor samples of patients with VTE had a proinflammatory status and contained higher levels of fibrin and fibrin degradation products than samples of those without VTE.

Conclusion

This case–control study provides a proof‐of‐principle that tumor gene expression can discriminate between cancer patients with low and high risks of VTE. These findings may help to further unravel the pathogenesis of cancer‐related VTE. The identified genes could potentially be used as candidate biomarkers to select high‐risk CRC patients for thromboprophylaxis.

     

Platelets kill bacteria by bridging innate and adaptive immunity via platelet factor 4 and FcγRIIA

Essentials

• Human platelets specifically interact with IgG opsonized bacteria through FcγRIIA.
• Platelet factor 4 (PF4) binds to polyanions (P) and undergoes a conformational change.
• Anti‐PF4/P IgG opsonizes PF4‐coated Gram‐positive and Gram‐negative bacteria.
• Platelets specifically kill E.coli opsonized with PF4 and human anti‐PF4/P IgG.

Summary

Background

Activated platelets release the chemokine platelet factor 4 (PF4) stored in their granules. PF4 binds to polyanions (P) on bacteria, undergoes a conformational change and exposes neoepitopes. These neoepitopes induce production of anti‐PF4/P antibodies. As PF4 binds to a variety of bacteria, anti‐PF4/P IgG can bind and opsonize several bacterial species.

Objective

Here we investigated whether platelets are able to kill bacteria directly after recognizing anti‐PF4/P IgG opsonized bacteria in the presence of PF4 via their FcγRIIA.

Methods

Using platelet‐bacteria suspension co‐culture experiments and micropatterns with immobilized viable bacteria, in combination with pharmacological inhibitors and human anti‐ PF4/P IgG we analyzed the role of platelet‐mediated killing of bacteria.

Results

In the presence of PF4, human anti‐PF4/P IgG and platelets, E. coli killing (> 50%) with colony forming units (CFU mL^?1) 0.71 × 10⁴ ± 0.19 was observed compared with controls incubated only with anti‐PF4/P IgG (CFU mL^?1 3.4 × 10⁴ ± 0.38). Blocking of platelet FcγRIIA using mAb IV.3 (CFU mL^?1 2.5 × 10⁴ ± 0.45), or integrin αIIbβ3 (CFU mL^?1 2.26 × 10⁴ ± 0.31), or disruption of cytoskeletal functions (CFU mL^?1 2.7 × 10⁴ ± 0.4) markedly reduced E. coli killing by this mechanism. Our observation of E. coli killing by platelets on micropatterned arrays is compatible with the model that platelets kill bacteria by covering them, actively concentrating them into the area under their granulomere and then releasing antimicrobial substances of platelet α‐granules site directed towards bacteria.

Conclusion

These findings collectively indicate that by bridging of innate and adaptive immune mechanisms, platelets and anti‐PF4/polyanion antibodies cooperate in an antibacterial host response.

     

Aspirin withdrawal in patients treated with ticagrelor presenting with non‐ST elevation myocardial infarction

Essentials

• Strong P2Y₁₂ blockade may cause platelet inhibition that is only minimally enhanced by aspirin.
• We evaluated aspirin withdrawal on platelet reactivity in ticagrelor treated patients.
• Aspirin withdrawal resulted in increased platelet reactivity to arachidonic acid.
• Aspirin withdrawal caused little difference in adenosine diphosphate‐induced platelet aggregation.

Summary

Background

Recent studies have shown that the thromboxane A₂‐dependent pathway is dependent on the ADP–P2Y₁₂ pathway, and that strong P2Y₁₂ receptor blockade alone causes inhibition of platelet aggregation that is minimally enhanced by aspirin. Data from the PLATO trial suggested that, among ticagrelor‐treated patients, high‐dose versus low‐dose (< 100 mg day^?1) aspirin is associated with an increased risk fof ischemic events.

Objectives

To evaluate the impact of aspirin withdrawal on platelet reactivity in acute coronary syndrome (ACS) patients treated with a potent P2Y₁₂ blocker.

Patients/Methods

This was a current prospective, randomized, placebo‐controlled, double‐blind, cross‐over study. The study population comprised 22 consecutive ACS patients who underwent percutaneous coronary intervention and were treated with aspirin (100 mg day^?1) and ticagrelor. Thirty days post‐ACS, open‐label aspirin was stopped, and patients were randomized to either blinded aspirin or placebo for 2 weeks, with each patient crossing over to the other arm for an additional 2 weeks. Platelet reactivity to arachidonic acid and ADP determined with light‐transmission aggregometry (LTA) and VerifyNow was evaluated at baseline, and 2 weeks and 4 weeks later.

Results

Aspirin withdrawal resulted in an increase in arachidonic‐acid induced platelet reactivity as determined with both LTA (77.0% ± 11.3% versus 20.8% ± 4.4%) and VerifyNow (607.7 ± 10.6 aspirin reaction units [ARU] versus 408.5 ± 14.4 ARU). Platelet response to ADP, as determined with both LTA and VerifyNow, did not differ with either aspirin or placebo (32.9% ± 2.6% versus 35.8% ± 3.6%, and 33.5 ± 6.4 P2Y₁₂ reaction units (PRU) versus 29.6 ± 5.7 PRU, respectively).

Conclusions

Aspirin withdrawal early post‐ACS results in increased platelet reactivity in response to arachidonic acid, despite concomitant treatment with the potent P2Y₁₂ blocker ticagrelor.

     

Human platelets express endothelial protein C receptor,which can be utilized to enhance localization of factor VIIa activity

Essentials

• Factor VIIa binds activated platelets to promote hemostasis in hemophilia patients with inhibitors.
• The interactions and sites responsible for platelet‐FVIIa binding are not fully understood.
• Endothelial cell protein C receptor (EPCR) is expressed on activated human platelets.
• EPCR binding enhances the efficacy of a FVIIa variant and could impact design of new therapeutics.

Summary

Background

High‐dose factor VIIa (FVIIa) is routinely used as an effective bypassing agent to treat hemophilia patients with inhibitory antibodies that compromise factor replacement. However, the mechanism by which FVIIa binds activated platelets to promote hemostasis is not fully understood. FVIIa‐DVQ is an analog of FVIIa with enhanced tissue factor (TF)‐independent activity and hemostatic efficacy relative to FVIIa. Our previous studies have shown that FVIIa‐DVQ exhibits greater platelet binding, thereby suggesting that features in addition to lipid composition contribute to platelet–FVIIa interactions.

Objectives

Endothelial cell protein C receptor (EPCR) also functions as a receptor for FVIIa on endothelial cells. We therefore hypothesized that an interaction with EPCR might play a role in platelet–FVIIa binding.

Methods/results

In the present study, we used flow cytometric analyses to show that platelet binding of both FVIIa and FVIIa‐DVQ is partially inhibited in the presence of excess protein C or an anti‐EPCR antibody. This decreased binding results in a corresponding decrease in the activity of both molecules in FXa and thrombin generation assays. Enhanced binding to EPCR was sufficient to account for the increased platelet binding of FVIIa‐DVQ compared with wild‐type FVIIa. As EPCR protein expression has not previously been shown in platelets, we confirmed the presence of EPCR in platelets using immunofluorescence, flow cytometry, immunoprecipitation, and mass spectrometry.

Conclusions

This work represents the first demonstration that human platelets express EPCR and suggests that modulation of EPCR binding could be utilized to enhance the hemostatic efficacy of rationally designed FVIIa analogs.

     

Metabolomic analysis of 92 pulmonary embolism patients from a nested case–control study identifies metabolites associated with adverse clinical outcomes

Essentials

• Risk‐stratification often fails to predict clinical deterioration in pulmonary embolism (PE).
• First‐ever high‐throughput metabolomics analysis of risk‐stratified PE patients.
• Changes in circulating metabolites reflect a compromised energy metabolism in PE.
• Metabolites play a key role in the pathophysiology and risk stratification of PE.

Summary

Background

Patients with acute pulmonary embolism (PE) exhibit wide variation in clinical presentation and outcomes. Our understanding of the pathophysiologic mechanisms differentiating low‐risk and high‐risk PE is limited, so current risk‐stratification efforts often fail to predict clinical deterioration and are insufficient to guide management.

Objectives

To improve our understanding of the physiology differentiating low‐risk from high‐risk PE, we conducted the first‐ever high‐throughput metabolomics analysis (843 named metabolites) comparing PE patients across risk strata within a nested case–control study.

Patients/methods

We enrolled 92 patients diagnosed with acute PE and collected plasma within 24 h of PE diagnosis. We used linear regression and pathway analysis to identify metabolites and pathways associated with PE risk‐strata.

Results

When we compared 46 low‐risk with 46 intermediate/high‐risk PEs, 50 metabolites were significantly different after multiple testing correction. These metabolites were enriched in the following pathways: tricarboxylic acid (TCA) cycle, fatty acid metabolism (acyl carnitine) and purine metabolism, (hypo)xanthine/inosine containing. Additionally, energy, nucleotide and amino acid pathways were downregulated in intermediate/high‐risk PE patients. When we compared 28 intermediate‐risk with 18 high‐risk PE patients, 41 metabolites differed at a nominal P‐value level. These metabolites were enriched in fatty acid metabolism (acyl cholines), and hemoglobin and porphyrin metabolism.

Conclusion

Our results suggest that high‐throughput metabolomics can provide insight into the pathophysiology of PE. Specifically, changes in circulating metabolites reflect compromised energy metabolism in intermediate/high‐risk PE patients. These findings demonstrate the important role metabolites play in the pathophysiology of PE and highlight metabolomics as a potential tool for risk stratification of PE.

     

Platelets contribute to the initiation of colitis‐associated cancer by promoting immunosuppression

Essentials

• Inflammation plays a key role in the development of colorectal cancer.
• Understanding mechanisms of cancer initiation might reveal new anticancer preventive strategy.
• Hyperactive platelets promote tumor formation by fostering immune evasion of cancer.
• Platelet inhibition by clopidogrel prevents carcinogenesis by restoring antitumor immunity.

Summary

Background

Clinical and experimental evidence support a role for inflammation in the development of colorectal cancer, although the mechanisms are not fully understood. Beyond thrombosis and hemostasis, platelets are key actors in inflammation; they have also been shown to be involved in cancer. However, whether platelets participate in the link between inflammation and cancer is unknown.

Objective

To investigate the contribution of platelets and platelet‐derived proteins to inflammation‐elicited colorectal tumor development.

Methods

We used a clinically relevant mouse model of colitis‐associated cancer. Platelet secretion and platelet reactivity to thrombin were assessed at each stage of carcinogenesis. We conducted an unbiased proteomic analysis of releasates of platelets isolated at the pretumoral stage to identify soluble factors that might act on tumor development. Plasma levels of the identified proteins were measured during the course of carcinogenesis. We then treated the mice with clopidogrel to efficiently inhibit platelet release reaction.

Results

At the pretumoral stage, hyperactive platelets constituted a major source of circulating protumoral serum amyloid A (SAA) proteins. Clopidogrel prevented the early elevation of the plasma SAA protein level, decreased colitis severity, and delayed the formation of dysplastic lesions and adenocarcinoma. Platelet inhibition hindered the expansion and function of immunosuppressive myeloid cells, as well as their infiltration into tumors, but increased the number of tissue CD8⁺ T cells. Platelets and releasates of platelets from mice with cancer were both able to polarize myeloid cells towards an immunosuppressive phenotype.

Conclusions

Thus, platelets promote the initiation of colitis‐associated cancer by enhancing myeloid cell‐dependent immunosuppression. Antiplatelet agents may help to prevent inflammation‐elicited carcinogenesis by restoring antitumor immunity.

     

Standardization of extracellular vesicle measurements by flow cytometry through vesicle diameter approximation

Essentials

• Platelet extracellular vesicles (EVs) concentrations measured by flow cytometers are incomparable.
• A model is applied to convert ambiguous scatter units to EV diameter in nanometer.
• Most included flow cytometers lack the sensitivity to detect EVs of 600 nm and smaller.
• The model outperforms polystyrene beads for comparability of platelet EV concentrations.

Summary

Background

Detection of extracellular vesicles (EVs) by flow cytometry has poor interlaboratory comparability, owing to differences in flow cytometer (FCM) sensitivity. Previous workshops distributed polystyrene beads to set a scatter‐based diameter gate in order to improve the comparability of EV concentration measurements. However, polystyrene beads provide limited insights into the diameter of detected EVs.

Objectives

To evaluate gates based on the estimated diameter of EVs instead of beads.

Methods

A calibration bead mixture and platelet EV samples were distributed to 33 participants. Beads and a light scattering model were used to set EV diameter gates in order to measure the concentration of CD61–phycoerythrin‐positive platelet EVs.

Results

Of the 46 evaluated FCMs, 21 FCMs detected the 600–1200‐nm EV diameter gate. The 1200–3000‐nm EV diameter gate was detected by 31 FCMs, with a measured EV concentration interlaboratory variability of 81% as compared with 139% with the bead diameter gate. Part of the variation in both approaches is caused by precipitation in some of the provided platelet EV samples. Flow rate calibration proved essential because systems configured to 60 μL min^?1 differed six‐fold in measured flow rates between instruments. Conclusions EV diameter gates improve the interlaboratory variability as compared with previous approaches. Of the evaluated FCMs, 24% could not detect 400‐nm polystyrene beads, and such instruments have limited utility for EV research. Finally, considerable differences were observed in sensitivity between optically similar instruments, indicating that maintenance and training affect the sensitivity.

     

Polymorphism in dural arteriovenous fistula: matrix metalloproteinase‐2‐1306 C/T as a potential risk factor for sinus thrombosis

Essentials

• Sinus thrombosis may play a crucial role in development of dural arteriovenous fistula (DAVF).
• Little is known about the association between gene polymorphism and the development of DAVF.
• MMP‐2‐1306 C/T showed a higher prevalence rate in DAVF cases with sinus thrombosis.
• MMP‐2‐1306C/T polymorphism is likely a potential risk factor for sinus thrombosis in DAVF.

Summary

Background

Dural arteriovenous fistula (DAVF) is a rare but important cerebrovascular disorder in adults. Little is known about the molecular genetic pathogenesis underlying DAVF development.

Objectives

To investigate the associations of gene polymorphisms and DAVF.

Materials and Methods

By the use of real‐time PCR genotyping, seven single‐nucleotide polymorphisms (SNPs) of angiogenesis‐related genes were analyzed in 72 DAVF patients. Pertinent clinical and imaging data were subgrouped on the basis of location (cavernous sinus versus lateral sinus), lesions (single versus multiple), cerebral venous reflux (CVR) grading (Borden I versus Borden II/III), and sinus thrombosis (with versus without).

Results

We found that individuals carrying the polymorphic allele of matrix metalloproteinase (MMP)‐2‐1306 C/T (rs243865) had a significantly increased risk of sinus thrombosis in DAVF (odds ratio 6.2; 95% confidence interval 1.7–22.9). There was a weak difference in associations of tissue inhibitor of metalloproteinase (TIMP)‐2 (rs2277698) gene polymorphism and DAVF patients subgrouped by CVR grading.

Conclusions

These preliminary results indicate that MMP‐2‐1306 C/T, but not MMP‐9, TIMP‐1, TIMP‐2, and vascular endothelial growth factor A SNP variants, is a risk factor for the development of sinus thrombosis in DAVF patients.

     

Functional characterization of recombinant snake venom rhodocytin: rhodocytin mutant blocks CLEC‐2/podoplanin‐dependent platelet aggregation and lung metastasis

Essentials

• We generated recombinant rhodocytin that could aggregate platelets via CLEC‐2.
• Recombinant wild‐type rhodocytin formed heterooctamer with four α‐ and β‐subunits.
• Asp 4 in α‐subunit of rhodocytin was required for binding to CLEC‐2.
• Inhibitory mutant of rhodocytin blocked podoplanin‐dependent hematogenous metastasis.

Summary

Background

Rhodocytin, a disulfide‐linked heterodimeric C‐type lectin from Calloselasma rhodostoma consisting of α‐subunits and β‐subunits, induces platelet aggregation through C‐type lectin‐like receptor 2 (CLEC‐2). CLEC‐2 is a physiological binding partner of podoplanin (PDPN), which is expressed on some tumor cell types, and is involved in tumor cell‐induced platelet aggregation and tumor metastasis. Thus, modified rhodocytin may be a possible source of anti‐CLEC‐2 drugs for both antiplatelet and antimetastasis therapy. However, its molecular function has not been well characterized, because of the lack of recombinant rhodocytin that induces platelet aggregation.

Objective

To produce recombinant rhodocytin, in order to verify its function with mutagenesis, and to develop an anti‐CLEC‐2 drug based on the findings.

Methods

We used Chinese hamster ovary cells to express recombinant rhodocytin (wild‐type [WT] and mutant), which was analyzed for induction/inhibition of platelet aggregation with light transmission aggregometry, the formation of multimers with blue native PAGE, and binding to CLEC‐2 with flow cytometry. Finally, we investigated whether mutant rhodocytin could suppress PDPN‐induced metastasis in an experimental lung metastasis mouse model.

Results

Functional WT] rhodocytin (αWTβWT) was obtained by coexpression of both subunits. Asp4 in α‐subunits of rhodocytin was required for CLEC‐2 binding. αWTβWT formed a heterooctamer similarly to native rhodocytin. Moreover, an inhibitory mutant of rhodocytin (αWTβK53A/R56A), forming a heterotetramer, bound to CLEC‐2 without inducing platelet aggregation, and blocked CLEC‐2–PDPN interaction‐dependent platelet aggregation and experimental lung metastasis.

Conclusion

These findings provide molecular characterization information on rhodocytin, and suggest that mutant rhodocytin could be used as a therapeutic agent to target CLEC‐2.

     

Development of a clinical prediction model for the postthrombotic syndrome in a prospective cohort of patients with proximal deep vein thrombosis

Essentials

• We developed a prediction model for postthrombotic syndrome (PTS) after deep vein thrombosis (DVT).
• High risk predictors were iliac vein DVT, BMI>35 and moderate‐severe Villalta category.
• Patients with a score ≥4 had an odds ratio of 5.9 (95% CI 2.1‐16.6) for PTS.
• SOX‐PTS score may select DVT patients for close monitoring or aggressive strategies to treat DVT.

Summary

Background

Postthrombotic syndrome (PTS) is a chronic complication that develops in 20–50% of patients after deep vein thrombosis (DVT). Although individual risk factors for PTS have been characterized, the ability to predict which DVT patients are likely to develop PTS remains limited.

Objective

To develop a clinical prediction score for PTS in patients with DVT.

Methods

The derivation cohort consisted of participants in the SOX Trial, a randomized double‐blind placebo‐controlled trial of elastic compression stockings versus placebo stockings worn for 2 years after DVT to prevent PTS in patients with a first proximal DVT, enrolled in 24 community and tertiary‐care hospitals from 2004 to 2010. Multivariable logistic regression analysis of baseline characteristics was performed. The outcome was the occurrence of PTS, diagnosed starting from 6 months or later according to Ginsberg's criteria.

Results

Seven hundred and sixty‐two patients were included in the analysis. The median follow‐up was 728 days. The model includes three independent predictors, and has a range of possible scores from 0 to 5. High‐risk predictors were: index DVT in the iliac vein; body mass index of ≥ 35 kg m^?2; and moderate–severe Villalta severity category at DVT diagnosis. As compared with patients with a score of 0, those with a score of ≥ 4 had an odds ratio of 5.9 (95% confidence interval 2.1–16.6) for developing PTS.

Conclusions

To our knowledge, this is the first clinical prediction score for PTS. We identified three independent predictors that, when combined, predicted PTS risk after a first proximal DVT. The SOX‐PTS score requires external validation before it can be considered for clinical use.

     

Reversibility of platelet P2Y12 inhibition by platelet supplementation: ex vivo and in vitro comparisons of prasugrel,clopidogrel and ticagrelor

Essentials

• Successful outcome of platelet transfusion depends on specific antiplatelet therapy in use.
• We assessed if ticagrelor, clopidogrel or prasugrel impacts on donor platelet activity ex vivo.
• Ticagrelor and/or its active metabolite in plasma or bound to platelets can inhibit donor platelets.
• This might compromise the effectiveness of platelet transfusion therapy.

Summary

Background

Platelet transfusion is the conventional approach to restore platelet function during acute bleeds or surgery, but successful outcome depends on the specific antiplatelet therapy. Notably ticagrelor is associated with inadequate recovery of platelet function after platelet transfusion. We examined whether plasma and/or platelets from ticagrelor‐treated patients influence donor platelet function, in comparison with clopidogrel and prasugrel.

Methods

Platelet transfusion was mimicked ex vivo by mixing naïve donor platelet‐rich plasma (PRP) or gel‐filtered platelets (GFP) in defined proportions with PRP, plasma or GFP from cardiovascular patients receiving standard care including medication with prasugrel, clopidogrel or ticagrelor (n = 20 each). Blood was taken 4 h after the previous dose. HLA2/HLA28 haplotyping let us distinguish net (all platelet) and individual patient/donor platelet reactivity in mixtures of patient/donor platelets, measured by flow cytometry analysis of ADP‐induced fibrinogen binding and CD62P expression.

Results

ADP responsiveness of donor platelets was dramatically reduced by even low (10%) concentrations of PRP or plasma from ticagrelor‐treated patients. Clopidogrel and prasugrel were associated with more modest donor platelet inhibition. GFP from ticagrelor‐treated patients but not patients receiving clopidogrel or prasugrel also suppressed donor GFP function upon mixing, suggesting the transfer of ticagrelor from patient platelets to donor platelets. This transfer did not lead to recovery of ADP responsiveness of patient's platelets.

Conclusion

Collectively, these observations support the concept that ticagrelor and/or its active metabolite in plasma or bound to platelets can inhibit donor platelets, which might compromise the effectiveness of platelet transfusion therapy.