Blood coagulation and beyond: position paper from the fourth Maastricht consensus conference on thrombosis

The Fourth Maastricht Consensus Conference on Thrombosis included the following themes. Theme 1: The "coagulome" as a critical driver of cardiovascular disease. Blood coagulation proteins also play divergent roles in biology and pathophysiology, related to specific organs, including brain, heart, bone marrow, and kidney. Four investigators shared their views on these organ- specific topics. Theme 2: Novel mechanisms of thrombosis. Mechanisms linking factor XII to fibrin, including their structural and physical properties, contribute to thrombosis, which is also affected by variation in microbiome status. Virus infection-associated coagulopathies perturb the hemostatic balance resulting in thrombosis and/ or bleeding. Theme 3: How to limit bleeding risks: insights from translational studies. This theme included state-of- the- art methodology for exploring the contribution of genetic determinants of a bleeding diathesis; determination of polymorphisms in genes that control the rate of metabolism by the liver of P2Y12 inhibitors, to improve safety of antithrombotic therapy. Novel reversal agents for direct oral anticoagulants are discussed. Theme 4: Hemostasis in extracorporeal systems: the value and limitations of ex vivo models. Perfusion flow chamber and nanotechnology developments are developed for studying bleeding and thrombosis tendencies. Vascularized organoids are utilized for disease modeling and drug development studies. Strategies for tackling extracorporeal membrane oxygenation-associated coagulopathy are discussed. Theme 5: Clinical dilemmas in thrombosis and antithrombotic management. Plenary presentations addressed controversial areas, i. e., thrombophilia testing, thrombosis risk assessment in hemophilia, novel antiplatelet strategies, and clinically tested factor XI(a) inhibitors, both possibly with reduced bleeding risk. Finally, COVID- 19-associated coagulopathy is revisited.Nephrolog

Platelet P2Y12 receptors enhance signalling towards procoagulant activity and thrombin generation. A study with healthy subjects and patients at thrombotic risk

Activated platelets participate in arterial thrombosis by forming aggregates and potentiating the coagulation through exposure of procoagulant phosphatidylserine. The function of the two receptors for ADP, P2Y(1) and P2Y(12), is well-established in aggregation, but is incompletely understood in the platelet procoagulant response. We established that, in PRP from healthy subjects, ADP accelerated and potentiated tissue factor induced thrombin generation exclusively via stimulation of P2Y(12) and not via P2Y(1) receptors. The P2Y(12) receptors also mediated the potentiating effect of PAR-1 stimulation on thrombin generation. Furthermore, ADP enhanced in a P2Y(12)-dependent manner the Ca(2+) response induced by thrombin, which was either added externally or generated in-situ. This ADP effect was in part dependent of phosphoinositide 3-kinase and was paralleled by increased phosphatidylserine exposure. In PRP from (young) patients with either stroke or type-II diabetes, platelet-dependent thrombin generation was similarly enhanced byADP or SFLLRN as in healthy subjects. In PRP from stroke patients of older age, the P2Y(12)-mediated contribution to thrombin generation was variably reduced by two weeks of clopidogrel medication. Remaining P2Y(12) activity after medication correlated with remaining P2Y(12)-dependent P-selectin exposure, i.e. Ca(2+)-dependent secretion, likely due to incomplete antagonism of P2Y(12) receptors. Together, these results indicate that physiological platelet agonists amplify phosphatidylserine exposure and subsequent thrombin generation by release of ADP and P2Y(12)-receptor stimulation. This P2Y(12) response is accomplished by a novel Ca(2+) signalling pathway. It is similarly active in platelets from control subjects and patients at thrombotic risk. Finally, the thrombogram method is useful for measuring incomplete P2Y(12) inhibition with clopidogrel

Effects of Platelet Agonists and Priming on the Formation of Platelet Populations

Platelets from healthy donors display heterogeneity in responsiveness to agonists. The response thresholds of platelets are controlled by multiple bioactive molecules, acting as negatively or positively priming substances. Higher circulating levels of priming substances adenosine and succinate, as well as the occurrence of hypercoagulability, have been described for patients with ischaemic heart disease. Here, we present an improved methodology of flow cytometric analyses of platelet activation and the characterisation of platelet populations following activation and priming by automated clustering analysis.Platelets were treated with adenosine, succinate, or coagulated plasma before stimulation with CRP-XL, 2-MeSADP, or TRAP6 and labelled for activated integrin alpha (IIb) beta (3) (PAC1), CD62P, TLT1, CD63, and GPIX. The Super-Enhanced Dmax subtraction algorithm and 2% marker (quadrant) setting were applied to identify populations, which were further defined by state-of-the-art clustering techniques (tSNE, FlowSOM). Following activation, five platelet populations were identified: resting, aggregating (PAC1+), secreting (alpha- and dense-granules; CD62P+, TLT1+, CD63+), aggregating plus alpha -granule secreting (PAC1+, CD62P+, TLT1+), and fully active platelet populations. The type of agonist determined the distribution of platelet populations. Adenosine in a dose-dependent way suppressed the fraction of fully activated platelets (TRAP6>2-MeSADP>CRP-XL), whereas succinate and coagulated plasma increased this fraction (CRP-XL>TRAP6>2-MeSADP). Interestingly, a subset of platelets showed a constant response (aggregating, secreting, or aggregating plus alpha -granule secreting), which was hardly affected by the stimulus strength or priming substances

Multiparameter Evaluation of the Platelet-Inhibitory Effects of Tyrosine Kinase Inhibitors Used for Cancer Treatment

Current antiplatelet drugs for the treatment of arterial thrombosis often coincide with increased bleeding risk. Several tyrosine kinase inhibitors (TKIs) for cancer treatment inhibit platelet function, with minor reported bleeding symptoms. The aim of this study was to compare the antiplatelet properties of eight TKIs to explore their possible repurposing as antiplatelet drugs. Samples of whole blood, platelet-rich plasma (PRP), or isolated platelets from healthy donors were treated with TKI or the vehicle. Measurements of platelet aggregation, activation, intracellular calcium mobilization, and whole-blood thrombus formation under flow were performed. Dasatinib and sunitinib dose-dependently reduced collagen-induced aggregation in PRP and washed platelets; pazopanib, cabozantinib, and vatalanib inhibited this response in washed platelets only; and fostamatinib, axitinib, and lapatinib showed no/limited effects. Fostamatinib reduced thrombus formation by approximately 50% on collagen and other substrates. Pazopanib, sunitinib, dasatinib, axitinib, and vatalanib mildly reduced thrombus formation on collagen by 10-50%. Intracellular calcium responses in isolated platelets were inhibited by dasatinib (> 90%), fostamatinib (57%), sunitinib (77%), and pazopanib (82%). Upon glycoprotein-VI receptor stimulation, fostamatinib, cabozantinib, and vatalanib decreased highly activated platelet populations by approximately 15%, while increasing resting populations by 39%. In conclusion, the TKIs with the highest affinities for platelet-expressed molecular targets most strongly inhibited platelet functions. Dasatinib, fostamatinib, sunitinib, and pazopanib interfered in early collagen receptor-induced molecular-signaling compared with cabozantinib and vatalanib. Fostamatinib, sunitinib, pazopanib, and vatalanib may be promising for future evaluation as antiplatelet drugs.</p

Tyrosine Kinase Inhibitor Sunitinib Delays Platelet-Induced Coagulation: Additive Effects of Aspirin

Background Sunitinib is a multitarget tyrosine kinase inhibitor (TKI) used for cancer treatment. In platelets, sunitinib affects collagen-induced activation under noncoagulating conditions. We investigated (1) the effects of sunitinib on thrombus formation induced by other TK-dependent receptors, and (2) the effects under coagulating conditions. Cardiovascular disease is a comorbidity in cancer patients, resulting in possible aspirin treatment. Sunitinib and aspirin are associated with increased bleeding risk, and therefore we also investigated (3) the synergistic effects of these compounds on thrombus and fibrin formation.Methods Blood or isolated platelets from healthy volunteers or cancer patients were incubated with sunitinib and/or aspirin or vehicle. Platelet activation was determined by TK phosphorylation, flow cytometry, changes in [Ca (2+) ] (i) , aggregometry, and whole blood perfusion over multiple surfaces, including collagen with(out) tissue factor (TF) was performed.Results Sunitinib reduced thrombus formation and phosphatidylserine (PS) exposure under flow on collagen type I and III. Also, sunitinib inhibited glycoprotein VI-induced TK phosphorylation and Ca (2+) elevation. Upon TF-triggered coagulation, sunitinib decreased PS exposure and fibrin formation. In blood from cancer patients more pronounced effects of sunitinib were observed in lung and pancreatic as compared to neuroglioblastoma and other cancer types. Compared to sunitinib alone, sunitinib plus aspirin further reduced platelet aggregation, thrombus formation, and PS exposure on collagen under flow with(out) coagulation.Conclusion Sunitinib suppresses collagen-induced procoagulant activity and delays fibrin formation, which was aggravated by aspirin. Therefore, we urge for awareness of the combined antiplatelet effects of TKIs with aspirin, as this may result in increased risk of bleeding

Differential Impact of Cytochrome 2C19 Allelic Variants on Three Different Platelet Function Tests in Clopidogrel-Treated Patients

On-treatment platelet reactivity in clopidogrel-treated patients can be measured with several platelet function tests (PFTs). However, the agreement between different PFTs is only slight to moderate. Polymorphisms of the CYP2C19 gene have an impact on the metabolization of clopidogrel and, thereby, have an impact on on-treatment platelet reactivity. The aim of the current study is to evaluate the differential effects of the CYP2C19 genotype on three different PFTs. Methods: From a prospective cohort study, we included patients treated with clopidogrel following percutaneous coronary intervention (PCI). One month after PCI, we simultaneously performed three different PFTs; light transmission aggregometry (LTA), VerifyNow P2Y12, and Multiplate. In whole EDTA blood, genotyping of the CYP2C19 polymorphisms was performed. Results: We included 308 patients treated with clopidogrel in combination with aspirin (69.5%) and/or anticoagulants (33.8%) and, based on CYP2C19 genotyping, classified them as either extensive (36.4%), rapid (34.7%), intermediate (26.0%), or poor metabolizers (2.9%). On-treatment platelet reactivity as measured by LTA and VerifyNow is significantly affected by CYP2C19 metabolizer status (p < 0.01); as metabolizer status changes from rapid, via extensive and intermediate, to poor, the mean platelet reactivity increases accordingly (p < 0.01). On the contrary, for Multiplate, no such ordering of metabolizer groups was found (p = 0.10). Conclusions: For VerifyNow and LTA, the on-treatment platelet reactivity in clopidogrel-treated patients correlates well with the underlying CYP2C19 polymorphism. For Multiplate, no major effect of genetic background could be shown, and effects of other (patient-related) variables prevail. Thus, besides differences in test principles and the influence of patient-related factors, the disagreement between PFTs is partly explained by differential effects of the CYP2C19 genotype