Pharmacological Blockade of Glycoprotein VI Promotes Thrombus Disaggregation in the Absence of Thrombin

Objective: Atherothrombosis occurs upon rupture of an atherosclerotic plaque and leads to the formation of a mural thrombus. Computational fluid dynamics and numerical models indicated that the mechanical stress applied to a thrombus increases dramatically as a thrombus grows, and that strong inter-platelet interactions are essential to maintain its stability. We investigated whether GPVI (glycoprotein VI)-mediated platelet activation helps to maintain thrombus stability by using real-time video-microscopy. Approach and Results: We showed that GPVI blockade with 2 distinct Fab fragments promoted efficient disaggregation of human thrombi preformed on collagen or on human atherosclerotic plaque material in the absence of thrombin. ACT017-induced disaggregation was achieved under arterial blood flow conditions, and its effect increased with wall shear rate. GPVI regulated platelet activation within a growing thrombus as evidenced by the loss in thrombus contraction when GPVI was blocked, and the absence of the disaggregating effect of an anti-GPVI agent when the thrombi were fully activated with soluble agonists. The GPVI-dependent thrombus stabilizing effect was further supported by the fact that inhibition of any of the 4 key immunoreceptor tyrosine-based motif signalling molecules, src-kinases, Syk, PI3Kβ, or phospholipase C, resulted in kinetics of thrombus disaggregation similar to ACT017. The absence of ACT017-induced disaggregation of thrombi from 2 afibrinogenemic patients suggests that the role of GPVI requires interaction with fibrinogen. Finally, platelet disaggregation of fibrin-rich thrombi was also promoted by ACT017 in combination with r-tPA (recombinant tissue plasminogen activator). Conclusions: This work identifies an unrecognized role for GPVI in maintaining thrombus stability and suggests that targeting GPVI could dissolve platelet aggregates with a poor fibrin content.

Comparison of acetylsalicylic acid and clopidogrel non-responsiveness assessed by light transmittance aggregometry and PFA-100® in patients undergoing neuroendovascular procedures

ObjectivesDual platelet inhibition is commonly used for prevention of cardiovascular events in patients undergoing neuroendovascular procedures. Non-responsiveness to platelet inhibitors may be associated with adverse outcomes. The aim of this study was to evaluate the reliability of the platelet function analyzer PFA-100® in comparison to light transmittance aggregometry (LTA) for monitoring clopidogrel and acetylsalicylic acid (ASA) non-responsiveness in a cohort of patients treated for intracranial aneurysm or cranial artery stenosis.MethodsNon-responsiveness to clopidogrel and ASA was assessed by LTA using adenosine diphosphate (ADP) and arachidonic acid and by PFA-100® with the ADP/prostaglandin E1 (PGE1) and collagen/epinephrine cartridges, respectively.ResultsA total of 203 patients (145 females; median age, 57 years) were analyzed. Agreement between the two tests was poor for clopidogrel non-responsiveness (ƙ=0.19) and not better than chance for ASA non-responsiveness (ƙ=0.01). Clopidogrel non-responsiveness by LTA and PFA-100® was associated with higher von Willebrand factor antigen and activity levels. ADP-induced platelet disaggregation was lower in patients with clopidogrel non-responsiveness as assessed by PFA-100®. Clopidogrel non-responsiveness by LTA was associated with a higher prevalence of diabetes and a higher body mass index (BMI). Adverse outcomes (death, thromboembolism, or in-stent thrombosis) occurred in 13% (n=26) of all patients independently of ASA and clopidogrel non-responsiveness as assessed by both devices.ConclusionsOur results show that LTA and PFA-100® are not interchangeable in the assessment of ASA and clopidogrel non-responsiveness in patients undergoing neuroendovascular interventions.

Synthetic Strategies for Dinucleotides Synthesis

Dinucleoside 5′,5′-polyphosphates (DNPs) are endogenous substances that play important intra- and extracellular roles in various biological processes, such as cell proliferation, regulation of enzymes, neurotransmission, platelet disaggregation and modulation of vascular tone. Various methodologies have been developed over the past fifty years to access these compounds, involving enzymatic processes or chemical procedures based either on P(III) or P(V) chemistry. Both solution-phase and solid-support strategies have been developed and are reported here. Recently, green chemistry approaches have emerged, offering attracting alternatives. This review outlines the main synthetic pathways for the preparation of dinucleoside 5′,5′-polyphosphates, focusing on pharmacologically relevant compounds, and highlighting recent advances.

Integrin-αIIbβ3-mediated outside-in signalling activates a negative feedback pathway to suppress platelet activation

SummaryIntegrin-αIIbβ3-mediated outside-in signalling is widely accepted as an amplifier of platelet activation; accumulating evidence suggests that outside-in signalling can, under certain conditions, also function as an inhibitor of platelet activation. The role of integrin-αIIbβ3-mediated outside-in signalling in platelet activation is disputable. We employed flow cytometry, aggregometry, immunoprecipitation, and immunoblotting to investigate the role of integrin-αIIbβ3-mediated outside-in signalling in platelet activation. Integrin αIIbβ3 inhibition enhances agonist-induced platelet ATP secretion. Human platelets lacking expression of αIIbβ3 exhibited more platelet ATP secretion than their wild-type counterparts. Moreover, integrin-αIIbβ3-mediated outside-in signals activate SHIP-1, which in turn mediates p-Akt dep-hosphorylation, leading to inactivation of PI3K/Akt signalling. Furthermore, 3AC (SHIP-1 inhibitor) inhibits platelet disaggregation, and promotes platelet ATP secretion. Upon ADP stimulation, Talin is recruited to αIIbβ3, and it is dissociated from αIIbβ3 when platelets disaggregate. In addition, treatment with RUC2, an inhibitor of αIIbβ3, which blocks αIIbβ3-mediated outside-in signalling, can markedly prevent the dissociation of talin from integrin. SHIP1 Inhibitor 3AC inhibits the dissociation of talin from integrin-β3. These results suggest that integrin-αIIbβ3-mediated outside-in signalling can serve as a brake to restrict unnecessary platelet activation by activated SHIP-1, which mediated the disassociation of talin from β3, leading to integrin inactivation and blocking of PI3K/Akt signalling to restrict platelet ATP secretion.

Disaggregation Signals Initiated by Ligand Binding and Platelet Aggregation

Abstract 2196 Accumulating evidence suggests that activation of the major platelet integrin, αIIbβ3, and subsequent platelet aggregation are intrinsically dynamic and reversible processes, especially when platelets are activated by low-dose agonists like ADP. When aggregation is not robust enough to activate feed-forward autocrine amplification pathways, platelet integrins revert to their inactivated state and fibrinogen dissociates, resulting in platelet disaggregation. Failure to disaggregate following low-dose agonist stimulation has been known for many years to correlate with occlusive arterial disease and diabetes mellitus, however the mechanisms by which integrins become inactivated leading to dissociation of fibrinogen is not well understood. Because phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling is known to be required to maintain integrins in an activated state and to support the formation of stable platelet aggregates, we examined whether the PI3K/Akt signaling might be actively reversed when platelets aggregate in response to ADP. We found that Akt becomes completely dephosphorylated in an aggregation-dependent manner. SHIP-1, a negative regulator of PI3K signaling, also becomes phosphorylated and activated during the early stages of platelet aggregation. These results suggest that fibrinogen binding and platelet aggregation initiate negative feedback through the PI3K/Akt signaling pathway to control unnecessary platelet aggregation responses. To gain further insight into the mechanism of platelet disaggregation, we examined the dynamic interaction of αIIbβ3 with talin – a cytosolic protein whose association with the integrin β3 cytoplasmic domain is required for integrin activation. We found that talin translocates to αIIbβ3 upon ADP stimulation, but dissociates from it during the process of platelet disaggregation. Blocking ADP-induced platelet aggregation with RGDW inhibited dissociation of talin from the integrin, again supporting the notion that that platelet aggregation initiates negative, as well as positive, signals to regulate the extent of agonist-induced platelet aggregation. Finally, we found that platelets missing PLCγ2 or FcγRIIa, each of which plays a positive role downstream of αIIbβ3-mediated outside-in signaling, are less prone to disaggregate following low-dose agonist stimulation than are their wild-type counterparts, although neither of them plays a role in ADP-induced platelet aggregation. Taken together, these data demonstrate that platelet aggregation and integrin αIIbβ3-mediated outside-in signaling not only initiates signals that amplify integrin activation and platelet aggregation, but also guards against inadvertent activation by shifting activated integrin αIIbβ3 back to its resting state, triggering platelet contraction and leading to dissociation of platelet aggregates. Disclosures: No relevant conflicts of interest to declare.

Hemostasis effects of various glucose-lowering medications and angiopathy prevention

Aim. To assess hemostasis effects of various glucose-lowering medications (GLM), such as sulphanilamides, biguanides, and insulin, in regard to micro- and microangiopathy progression over 5 years in patients with Type 2 diabetes mellitus (DM-2). Material and methods. The study included 72 patients with DM-2 (47 women, 25 men; median age 54,0 years (49,0-59,0 years); mean DM-2 duration 4,0 years (0,5-8,0 years)), receiving one of the following GLMs for 5 years: glibenclamide, gliclazide, metformin, insulin, or the combination of glibenclamide and metformin. Results. Various GLMs had different effects on platelet activity. In the gliclazide group, aggregant activity was decreased, compared to the glibenclamide and insulin groups. In the metformin group, aggregant activity and platelet disaggregation were similar to that in the gliclazide group. The microangiopathy progression over 5 years was related to GLM therapy, being minimal in the DM-2 patients receiving metformin, and maximal among participants administered glibenclamide. The microangiopathy progression was minimal in the metformin and gliclazide groups, and maximal — in the glibenclamide group. Conclusion. Carbohydrate and lipid metabolism compensation is not the only condition of angiopathy prevention. The latter should be based on the complex intervention, aimed at the complete metabolic compensation and hemostasis balance. The need for taking pleiotropic activity of specific agents into consideration when choosing GLM therapy is emphasized by protective effects of metformin (micro- and microangiopathy prevention) and gliclazide (microangiopathy prevention), but not glibenclamide.

Metabolic and hemostatic characteristics of patients with type 2 diabetes mellitus differing in the severity of angiopathy

The objective of the present study was to identify risk factors of developing vascular disorders in patients at different stages of type 2 diabetes mellitus (DM2) by comprehensive analysis of metabolic parameters, hemograms, thrombocytic and plasma hemostasis. The study involved 75 patients (22 men and 53 women of mean age 57,3±9,7 years) having angiopathies of different severity. The data obtained confirmed the presence of risk factors of vascular pathology in different phases of DM2. All the examined patients including those without angiopathies in the early period of diabetes showed triglyceridemia, cholesterolemia, enhanced platelet aggregation activity, and shortened activated partial thromboplastin time. Patients with diabetic nephropathy at the stage of microalbuminuria and with non-proliferative retinopathy were distinct from the remaining ones in that they had significantly higher blood alpha-2 globulin and fibrinogen levels. Diabetic patients with micro- and macrovascular problems were characterized by marked dysproteinemia and abnormal platelet disaggregation.