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.

     

Assessment of whole blood coagulation with a microfluidic dielectric sensor

Essentials

• ClotChip is a novel microsensor for comprehensive assessment of ex vivo hemostasis.
• Clinical samples show high sensitivity to detecting the entire hemostatic process.
• ClotChip readout exhibits distinct information on coagulation factor and platelet abnormalities.
• ClotChip has potential as a point‐of‐care platform for comprehensive hemostatic analysis.

Summary

Background

Rapid point‐of‐care (POC) assessment of hemostasis is clinically important in patients with a variety of coagulation factor and platelet defects who have bleeding disorders.

Objective

To evaluate a novel dielectric microsensor, termed ClotChip, which is based on the electrical technique of dielectric spectroscopy for rapid, comprehensive assessment of whole blood coagulation.

Methods

The ClotChip is a three‐dimensional, parallel‐plate, capacitive sensor integrated into a single‐use microfluidic channel with miniscule sample volume (< 10 μL). The ClotChip readout is defined as the temporal variation in the real part of dielectric permittivity of whole blood at 1 MHz.

Results

The ClotChip readout exhibits two distinct parameters, namely, the time to reach a permittivity peak (T_peak) and the maximum change in permittivity after the peak (Δε_r,max), which are, respectively, sensitive towards detecting non‐cellular (i.e. coagulation factor) and cellular (i.e. platelet) abnormalities in the hemostatic process. We evaluated the performance of ClotChip using clinical blood samples from 15 healthy volunteers and 12 patients suffering from coagulation defects. The ClotChip T_peak parameter exhibited superior sensitivity at distinguishing coagulation disorders as compared with conventional screening coagulation tests. Moreover, the ClotChip Δε_r,max parameter detected platelet function inhibition induced by aspirin and exhibited strong positive correlation with light transmission aggregometry.

Conclusions

This study demonstrates that ClotChip assesses multiple aspects of the hemostatic process in whole blood on a single disposable cartridge, highlighting its potential as a POC platform for rapid, comprehensive hemostatic analysis.

     

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.

     

The small‐molecule MERTK inhibitor UNC2025 decreases platelet activation and prevents thrombosis

Essentials

• Signaling by Gas6 through Tyro3/Axl/Mer receptors is essential for stable platelet aggregation.
• UNC2025 is a small molecule inhibitor of the Mer tyrosine kinase.
• UNC2025 decreases platelet activation in vitro and thrombus formation in vivo.
• UNC2025's anti‐platelet effect is synergistic with inhibition of the ADP receptor, P2Y₁₂.

Summary

Background

Growth arrest‐specific protein 6 signals through the TAM (TYRO‐3–AXL–MERTK) receptor family, mediating platelet activation and thrombus formation via activation of the aggregate‐stabilizing α_IIbβ₃ integrin.

Objective

To describe the antithrombotic effects mediated by UNC2025, a small‐molecule MERTK tyrosine kinase inhibitor.

Methods

MERTK phosphorylation and downstream signaling were assessed by immunoblotting. Light transmission aggregometry, flow cytometry and microfluidic analysis were used to evaluate the impact of MERTK inhibition on platelet activation and stability of aggregates in vitro. The effects of MERTK inhibition on arterial and venous thrombosis, platelet accumulation at microvascular injury sites and tail bleeding times were determined with murine models. The effects of combined treatment with ADP–P2Y_1&12 pathway antagonists and UNC2025 were also evaluated.

Results and Conclusions

Treatment with UNC2025 inhibited MERTK phosphorylation and downstream activation of AKT and SRC, decreased platelet activation, and protected animals from pulmonary embolism and arterial thrombosis without increasing bleeding times. The antiplatelet effect of UNC2025 was enhanced in combination with ADP–P2Y_1&12 pathway antagonists, and a greater than additive effect was observed when these two agents with different mechanisms of inhibition were coadministered. TAM kinase signaling represents a potential therapeutic target, as inhibition of this axis, especially in combination with ADP–P2Y pathway antagonism, mediates decreased platelet activation, aggregate stability, and thrombus formation, with less hemorrhagic potential than current treatment strategies. The data presented here also demonstrate antithrombotic activity mediated by UNC2025, a novel translational agent, and support the development of TAM kinase inhibitors for clinical applications.

     

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.

     

Thrombocytopenia in high‐risk patients with antiphospholipid syndrome

Essentials

• The prevalence of thrombocytopenia in patients with antiphospholipid syndrome is not well defined.
• We studied triple positive patients with antiphospholipid syndrome and its catastrophic variant.
• Prevalence of thrombocytopenia was 6% and 100% in patients who developed the catastrophic form.
• In triple positive patients thrombocytopenia is low and platelets drop during the catastrophic form.

Summary

Background

Thrombocytopenia is the most common non‐criteria hematological feature in patients with antiphospholipid syndrome (APS). This condition is more common in patients with catastrophic APS (CAPS).

Objectives

To evaluate the prevalence of thrombocytopenia in a large series of high‐risk patients with APS, and to assess the behavior of the platelet count during CAPS.

Methods/Patients

This was a cross‐sectional study in which we analyzed the platelet counts of a homogeneous group of high‐risk APS patients (triple‐positive). Six of these patients developed a catastrophic phase of the disease, and the platelet count was recorded before the acute phase, during the acute phase, and at recovery.

Results

The mean platelet count in 119 high‐risk triple‐positive patients was 210 × 10⁹ L^?1. With a cut‐off value for thrombocytopenia of 100 × 10⁹ L^?1, the prevalence of thrombocytopenia was 6% (seven patients). No difference between primary APS and secondary APS was found. In patients who suffered from CAPS, a significant decrease from the basal count (212 ± 51 × 10⁹ L^?1) to that at the time of diagnosis (60 ± 33 × 10⁹ L^?1) was observed. The platelet count became normal again at the time of complete remission (220 ± 57 × 10⁹ L^?1). A decrease in platelet count always preceded the full clinical picture.

Conclusions

This study shows that, in high‐risk APS patients, the prevalence of thrombocytopenia is low. A decrease in platelet count was observed in all of the patients who developed the catastrophic form of the disease. A decrease in platelet count in high‐risk APS patients should be considered a warning signal for disease progression to CAPS.

     

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.

     

Dabigatran versus vitamin k antagonist: an observational across‐cohort comparison in acute coronary syndrome patients with atrial fibrillation

Essentials

• Acute coronary syndrome (ACS) with atrial fibrillation (AF) is a therapeutic challenge.
• Dual and triple antithrombotic therapy showed a similar thrombotic risk in ACS patients with AF.
• The omission of aspirin during the first month did not increase the rate of ischemic events.
• Replacement of vitamin K antagonist by dabigatran leads to an increased thrombotic risk.

Summary

Background

Dual antithrombotic therapy comprising a vitamin K antagonist (VKA) plus clopidogrel reduces the incidence of major bleeding compared with triple therapy (VKA + clopidogrel + aspirin) in acute coronary syndrome (ACS) patients with atrial fibrillation (AF), with a similar thrombotic risk. The oral thrombin inhibitor dabigatran (150 mg twice a day) showed superiority over VKA in non‐valvular AF, but data supporting its use in AF patients presenting with ACS are limited.

Objective

We sought to evaluate the efficacy of dabigatran vs. VKA in the management of AF patients undergoing percutaneous coronary intervention for an ACS.

Methods

In this open‐label study, 133 consecutive patients received dabigatran plus clopidogrel. Another cohort of 133 patients treated with VKA plus clopidogrel was used as the control group.

Results

After propensity score adjustment, the cumulative incidence of major adverse cardiovascular events over 24 months was higher with dabigatran vs. VKA (adjusted hazard ratio, 2.28; 95% confidence interval, 1.46–3.56). Similar rates of major bleeding were found (adjusted hazard ratio, 1.17; 95% confidence interval, 0.46–2.96).

Conclusions

In AF patients presenting with ACS, replacement of VKA by dabigatran concurrently with clopidogrel is associated with an increased thrombotic risk, without a reduction in major bleeding.

     

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 .

     

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.

     

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.

     

Dual endothelin‐1 receptor antagonism attenuates platelet‐mediated derangements of blood coagulation in Eisenmenger syndrome

Essentials

• Eisenmenger syndrome is characterised by thrombotic and hemorrhagic risks of unclear aetiology.
• Calibrated automated thrombography was used to assess these coagulation derangements.
• Platelet activity supported abnormalities in procoagulant and anticoagulant pathway function.
• Endothelin‐1 antagonism appeared to ameliorate these derangements.

Summary

Aims

The mechanisms underlying the competing thrombotic and hemorrhagic risks in Eisenmenger syndrome are poorly understood. We aimed to characterize derangements of blood coagulation and to assess the effect of dual endothelin‐1 receptor antagonism in modulating hemostasis in this rare disorder.

Methods

In a 10‐month recruitment period at a tertiary cardiology referral center, during which time there were over 14 000 outpatient consultations, consecutive subjects with Eisenmenger syndrome being considered for macitentan therapy (n = 9) and healthy volunteers (n = 9) were recruited. Plasma thrombin generation in platelet‐rich and platelet‐poor plasma was assessed by calibrated automated thrombography prior to and following therapy.

Results

Median peak plasma thrombin generation was higher in platelet‐rich plasma obtained from Eisenmenger syndrome subjects relative to controls (median peak thrombin [25th–75th percentile]: 228.3 [206.5–258.6] nm vs. 169.9 [164.3–215.8] nm ), suggesting a critical mechanistic role for platelets in supporting abnormal hypercoagulability in Eisenmenger syndrome. Abnormal enhanced sensitivity to the anticoagulant activity of activated protein C was also observed in platelet‐rich plasma in Eisenmenger syndrome, suggesting that derangements of platelet activity may influence the activity of anticoagulant pathways in a manner that might promote bleeding in this disease state. Following 6 months of macitentan therapy, attenuations in the derangements in both procoagulant and anticoagulant pathways were observed.

Conclusions

Abnormal platelet activity contributes to derangements in procoagulant and anticoagulant pathways in Eisenmenger syndrome. Therapies targeting the underlying vascular pathology appear to ameliorate these derangements and may represent a novel strategy for the management of the competing prothrombotic and hemorrhagic tendencies in this disorder.

     

Platelets loaded with liposome‐encapsulated thrombin have increased coagulability

Essentials

• Platelet transfusions can have limited efficacy during hemorrhage associated with coagulopathy.
• Thrombin can be shielded by encapsulation into nanoliposomes and delivered to platelets ex vivo.
• Loading platelets with liposomal thrombin improved several aspects of platelet coagulability.
• Platelets loaded with liposomal thrombin can overcome some coagulopathic deficiencies in vitro.

Summary

Background

Platelets are integral to clot formation and are often transfused to stop or prevent bleeding. However, transfusions of platelets are not always effective, particularly in the most severe cases of hemorrhage. Nanoparticle systems have been developed to mimic platelets but inherently lack important aspects of platelet function, which limits their potential effectiveness.

Objectives

Increasing the natural coagulability of transfusable platelets could increase their efficacy during treatment of severe hemorrhage. Thrombin is a potent platelet agonist that currently cannot be used intravenously because of the risk of thrombosis. We hypothesized that delivery of thrombin to ex vivo platelets via liposomal encapsulation would enable transfusable platelets to become more coagulable in response to platelet agonists.

Methods

Thrombin was encapsulated into nanoliposomes and delivered to platelets ex vivo. Platelet coagulability was measured by monitoring platelet activation, clot contraction, clot time and clot stability in several in vitro assays. These parameters were also measured under conditions where coagulation is compromised, including during acidosis, antiplatelet drugs, hemophilia A and trauma‐induced coagulopathy.

Results

Liposomal thrombin was endocytosed and used by platelets ex vivo but was not secreted upon activation. These modified platelets became more sensitive and responsive to agonists and improved clotting time even under conditions that normally cause platelet dysfunction or have impaired coagulation.

Conclusions

Several aspects of platelet function were enhanced by ex vivo delivery of liposomal thrombin.

     

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.

     

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.

     

Carboxypeptidase U (CPU,carboxypeptidase B2, activated thrombin‐activatable fibrinolysis inhibitor) inhibition stimulates the fibrinolytic rate in different in vitro models

Essentials

• AZD9684 is a potent inhibitor of carboxypeptidase U (CPU, TAFIa, CPB2).
• The effect of AZD9684 on fibrinolysis was investigated in four in vitro systems.
• The CPU system also attenuates fibrinolysis in more advanced hemostatic systems.
• The size of the observed effect on fibrinolysis is dependent on the exact experimental conditions.

Summary

Background

Carboxypeptidase U (CPU, carboxypeptidase B2, activated thrombin‐activatable fibrinolysis inhibitor) is a basic carboxypeptidase that attenuates fibrinolysis. This characteristic has raised interest in the scientific community and pharmaceutical industry for the development of inhibitors as profibrinolytic agents.

Objectives

Little is known about the contribution of CPU to clot resistance in more advanced hemostatic models, which include blood cells and shear stress. The aim of this study was to evaluate the effects of the CPU system in in vitro systems for fibrinolysis with different grades of complexity.

Methods

The contribution of the CPU system was evaluated in the following systems: (i) plasma clot lysis; (ii) rotational thromboelastometry (ROTEM) in whole blood; (iii) front lysis with confocal microscopy in platelet‐free and platelet‐rich plasma; and (iv) a microfluidic system with whole blood under arterial shear stress. Experiments were carried out in the presence or absence of AZD9684, a specific CPU inhibitor.

Results

During plasma clot lysis, addition of AZD9684 resulted in 33% faster lysis. In ROTEM, the lysis onset time was decreased by 38%. For both clot lysis and ROTEM, an AZD9684 dose‐dependent response was observed. CPU inhibition in front lysis experiments resulted in 47% and 50% faster lysis for platelet‐free plasma and platelet‐rich plasma, respectively. Finally, a tendency for faster lysis was observed only in the microfluidic system when AZD9684 was added.

Conclusions

Overall, these experiments provide novel evidence that the CPU system can also modulate fibrinolysis in more advanced hemostatic systems. The extent of the effects appears to be dependent upon the exact experimental conditions.

     

Hollow organosilica beads as reference particles for optical detection of extracellular vesicles

Essentials

• Standardization of extracellular vesicle (EV) measurements by flow cytometry needs improvement.
• Hollow organosilica beads were prepared, characterized, and tested as reference particles.
• Light scattering properties of hollow beads resemble that of platelet‐derived EVs.
• Hollow beads are ideal reference particles to standardize scatter flow cytometry research on EVs.

Summary

Background

The concentration of extracellular vesicles (EVs) in body fluids is a promising biomarker for disease, and flow cytometry remains the clinically most applicable method to identify the cellular origin of single EVs in suspension. To compare concentration measurements of EVs between flow cytometers, solid polystyrene reference beads and EVs were distributed in the first ISTH‐organized interlaboratory comparison studies. The beads were used to set size gates based on light scatter, and the concentration of EVs was measured within the size gates. However, polystyrene beads lead to false size determination of EVs, owing to the mismatch in refractive index between beads and EVs. Moreover, polystyrene beads gate different EV sizes on different flow cytometers.

Objective

To prepare, characterize and test hollow organosilica beads (HOBs) as reference beads to set EV size gates in flow cytometry investigations.

Methods

HOBs were prepared with a hard template sol‐gel method, and extensively characterized for morphology, size, and colloidal stability. The applicability of HOBs as reference particles was investigated by flow cytometry with HOBs and platelet‐derived EVs.

Results

HOBs proved to be monodisperse with a homogeneous shell thickness. Two‐angle light‐scattering measurements by flow cytometry confirmed that HOBs have light‐scattering properties similar to those of platelet‐derived EVs.

Conclusions

Because the structure and light‐scattering properties HOBs resemble those of EVs, HOBs with a given size will gate EVs of the same size. Therefore, HOBs are ideal reference beads with which to standardize optical measurements of the EV concentration within a predefined size range.

     

TC21/RRas2 regulates glycoprotein VI–FcRγ‐mediated platelet activation and thrombus stability

Essentials

• RAS proteins are expressed in platelets but their functions are largely uncharacterized.
• TC21/RRas2 is required for glycoprotein VI‐induced platelet responses and for thrombus stability in vivo.
• TC21 regulates platelet aggregation by control of α_IIbβ₃ integrin activation, via crosstalk with Rap1b.
• This is the first indication of functional importance of a proto‐oncogenic RAS protein in platelets.

Summary

Background

Many RAS family small GTPases are expressed in platelets, including RAC, RHOA, RAP, and HRAS/NRAS/RRAS1, but most of their signaling and cellular functions remain poorly understood. Like RRAS1, TC21/RRAS2 reverses HRAS‐induced suppression of integrin activation in CHO cells. However, a role for TC21 in platelets has not been explored.

Objectives

To determine TC21 expression in platelets, TC21 activation in response to platelet agonists, and roles of TC21 in platelet function in in vitro and in vivo thrombosis.

Results

We demonstrate that TC21 is expressed in human and murine platelets, and is activated in response to agonists for the glycoprotein (GP) VI–FcRγ immunoreceptor tyrosine‐based activation motif (ITAM)‐containing collagen receptor, in an Src‐dependent manner. GPVI‐induced platelet aggregation, integrin α_IIbβ₃ activation, and α‐granule and dense granule secretion, as well as phosphorylation of Syk, phospholipase Cγ2, AKT, and extracellular signal‐regulated kinase, were inhibited in TC21‐deficient platelets ex vivo. In contrast, these responses were normal in TC21‐deficient platelets following stimulation with P2Y, protease‐activated receptor 4 and C‐type lectin receptor 2 receptor agonists, indicating that the function of TC21 in platelets is GPVI–FcRγ‐ITAM‐specific. TC21 was required for GPVI‐induced activation of Rap1b. TC21‐deficient mice did not show a significant delay in injury‐induced thrombosis as compared with wild‐type controls; however, thrombi were unstable. Hemostatic responses showed similar effects.

Conclusions

TC21 is essential for GPVI–FcRγ‐mediated platelet activation and for thrombus stability in vivo via control of Rap1b and integrins.

     

Reduced‐dose direct oral anticoagulants in the extended treatment of venous thromboembolism: a systematic review and meta‐analysis

Essentials

• In venous thromboembolism (VTE), benefits of extended treatment are balanced by bleeding risks.
• This is a meta‐analysis of reduced‐dose direct oral anticoagulants (DOACs) in extended treatment.
• Reduced‐dose DOACs are as effective as full anticoagulation with bleeding risks similar to placebo.
• Reduced‐dose DOACs are an attractive option for patients in the extended phase of VTE treatment.

Summary

Background

Extended‐duration anticoagulation is beneficial for preventing recurrent venous thromboembolism (VTE). Reduced‐dose direct oral anticoagulants (DOACs) may be preferable if they preserve efficacy and cause less bleeding. We conducted a systematic review and meta‐analysis of trials comparing reduced‐dose DOACs with full‐dose DOACs and aspirin or placebo in the extended phase of VTE treatment.

Methods

A literature search was conducted by use of the MEDLINE, EMBASE and CINAHL databases, supplemented by hand‐searching. One thousand three hundred and ninety‐nine titles were screened, with data from accepted studies being extracted by two independent reviewers. Major outcomes analyzed included recurrent VTE and major and clinically relevant non‐major bleeding events, presented as risk ratios (RRs) and 95% confidence intervals (CI).

Results

Two trials met the prespecified inclusion criteria. Data from 5847 patients were analyzed for efficacy outcomes, and from 5842 patients for safety outcomes. Reduced‐dose DOACs were as effective as full‐dose treatment in preventing recurrent VTE at 1 year (RR 1.12 [95% CI 0.67–1.87]), and more effective than aspirin or placebo (RR 0.26 [95% CI 0.14–0.46]). Rates of major or clinically relevant non‐major bleeding events were similar between patients receiving reduced‐dose DOACs and and those receiving aspirin or placebo (RR 1.19 [95% CI 0.81–1.77]). There was a trend towards less bleeding when reduced‐dose and full‐dose DOACs were compared (RR 0.74 [95% CI 0.52–1.05]).

Conclusions

Extended‐duration treatment of VTE with reduced‐dose DOACs may be as efficacious as full‐dose treatment, with rates of major bleeding being similar to those in patients receiving treatment with aspirin or placebo, but further long‐term studies are needed.

     

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.