Molecular Proteomics and Signalling of Human Platelets in Health and Disease

Platelets are small anucleate blood cells that play vital roles in haemostasis and thrombosis, besides other physiological and pathophysiological processes. These roles are tightly regulated by a complex network of signalling pathways. Mass spectrometry-based proteomic techniques are contributing not only to the identification and quantification of new platelet proteins, but also reveal post-translational modifications of these molecules, such as acetylation, glycosylation and phosphorylation. Moreover, target proteomic analysis of platelets can provide molecular biomarkers for genetic aberrations with established or non-established links to platelet dysfunctions. In this report, we review 67 reports regarding platelet proteomic analysis and signalling on a molecular base. Collectively, these provide detailed insight into the: (i) technical developments and limitations of the assessment of platelet (sub)proteomes; (ii) molecular protein changes upon ageing of platelets; (iii) complexity of platelet signalling pathways and functions in response to collagen, rhodocytin, thrombin, thromboxane A2 and ADP; (iv) proteomic effects of endothelial-derived mediators such as prostacyclin and the anti-platelet drug aspirin; and (v) molecular protein changes in platelets from patients with congenital disorders or cardiovascular disease. However, sample sizes are still low and the roles of differentially expressed proteins are often unknown. Based on the practical and technical possibilities and limitations, we provide a perspective for further improvements of the platelet proteomic field.

Do anti-platelet drugs improve duration of continuous renal replacement therapy? A retrospective cohort study in cardiac ICU patients

Objective: The objective of this study was to investigate the impact of anti-platelet drug/s on duration of continuous renal replacement therapy (CRRT) in those patients where anti-coagulants were not used due to certain contraindications and in cases where patients were on anti-platelet drugs and were given anti-coagulant during CRRT. Method: This single-center, retrospective cohort study was conducted using the medical records patients treated with CRRT in the cardiac ICU of the inpatient urban facility, located in North India. Data was collected from only those patients who received CRRT for the duration of at least 12 h. Patient’s in NAC group were not on any anti-platelet/s and did not receive anti-coagulant during CRRT. AC and AP group patients received anti-coagulant alone or were already on anti-platelet/s and did not receive anti-coagulant respectively while ACAP group patients were on anti-platelet drug/s and also received anti-coagulant during CRRT. Result: Patients in AC, AP, or ACAP group showed significantly ( p < 0.001) higher CRRT filter life compared to NAC group. The median CRRT filter life was significantly higher in the ACAP group compared to AC ( p < 0.05) and AP ( p < 0.001) groups. Conclusion: This study indicates that systemic anti-platelet therapy can provide additional support in critical patients undergoing CRRT even with or without anti-coagulant therapy. However, the increase in CRRT filter life was more profound in patients who were on anti-platelet/s and also received anti-coagulant drug/s during CRRT.

Cangrelor ameliorates CLP-induced pulmonary injury in sepsis by inhibiting GPR17

Background Sepsis is a common complication of severe wound injury and infection, with a very high mortality rate. The P2Y12 receptor inhibitor, cangrelor, is an antagonist anti-platelet drug. Methods In our study, we investigated the protective mechanisms of cangrelor in CLP-induced pulmonary injury in sepsis, using C57BL/6 mouse models. Results TdT-mediated dUTP Nick-End Labeling (TUNEL) and Masson staining showed that apoptosis and fibrosis in lungs were alleviated by cangrelor treatment. Cangrelor significantly promoted surface expression of CD40L on platelets and inhibited CLP-induced neutrophils in Bronchoalveolar lavage fluid (BALF) (p < 0.001). We also found that cangrelor decreased the inflammatory response in the CLP mouse model and inhibited the expression of inflammatory cytokines, IL-1β (p < 0.01), IL-6 (p < 0.05), and TNF-α (p < 0.001). Western blotting and RT-PCR showed that cangrelor inhibited the increased levels of G-protein-coupled receptor 17 (GPR17) induced by CLP (p < 0.001). Conclusion Our study indicated that cangrelor repressed the levels of GPR17, followed by a decrease in the inflammatory response and a rise of neutrophils in BALF, potentially reversing CLP-mediated pulmonary injury during sepsis.

Anti-platelet drug loaded targeted technologies for the effective treatment of Atherothrombosis

: Atherothrombosis is the result of direct interaction between atherosclerotic plaque and arterial thrombosis and is the most common type of cardiovascular disease. As a long term progressive disease, atherosclerosis frequently results in an acute atherothrombotic event through plaque rupture and platelet-rich thrombus formation. The pathophysiology of atherothrombosis involves cholesterol accumulation endothelial dysfunction, dyslipidemia, immuno-inflammatory and apoptotic aspects. Platelet activation and aggregation is the major cause for stroke because of its roles including thrombus, contributing to atherosclerotic plaque, and sealing off bleeding vessel. Platelet aggregates are associated with arterial blood pressure and cardiovascular ischemic events. Under normal physiological conditions when a blood vessel is damaged, the task of platelets within the circulation is to arrest the blood loss. Antiplatelet inhibit platelet function thereby decrease thrombus formation with complementary modes of action to prevent atherothrombosis. In the present scientific scenario, researchers throughout the world are focusing towards the development of novel drug delivery systems to enhance patient’s compliance. Immediate responding pharmaceutical formulations become an emerging trend in the pharmaceutical industries with better patient compliance. Here in the proposed review will focused towards the understanding on the molecular pathogenesis of atherothrombosis and recent novel formulation approaches for the treatment of atherothrombosis with special emphasis on commercial formulation and upcoming technologies.

Targeting Thymidine Phosphorylase with Tipiracil Hydrochloride is a Safe and Effective Antithrombotic Therapy

RationaleMost of the current anti-platelet drugs inhibit platelet function permanently and have systemic side effects, including thrombocytopenia and hemorrhage. We previously found that thymidine phosphorylase (TYMP), a platelet cytoplasmic protein, facilitates multiple agonist induced platelet activation and enhances thrombosis. A specific TYMP inhibitor, namely, tipiracil hydrochloride (TPI), has been approved by the U.S. Food and Drug Administration for clinical use as an auxiliary drug making it possible to be repositioned as an anti-platelet medicine.ObjectiveWe aimed to test the hypothesis that TPI is a novel and safe anti-platelet drug by examining its role in platelet activation and thrombosis using both in vitro and in vivo studies.Methods and ResultsBy co-expression of TYMP and Lyn or Lyn-SH3 domain tagged with glutathione S-transferase, we showed the direct evidence that TYMP binds to the SH3 domain in its partners. TYMP haplodeficiency is sufficient to inhibit thrombosis in vivo regardless of gender. TPI treatment rapidly inhibited collagen- and ADP-induced platelet aggregation, which copied the phenotype of TYMP deficient platelets. Under both normal and hyperlipidemic conditions, treating wild type (WT) mice with TPI via intraperitoneal injection, intravenous injection, or gavage feeding dramatically inhibited thrombosis without inducing significant bleeding. Even administered above the effective dose, TPI has a lower bleeding side effect compared to aspirin and clopidogrel. Most importantly, intravenously delivery of TPI alone or combined with tissue plasminogen activator dramatically inhibited the growth of developing thrombi. Dual administration of very low dose of aspirin and TPI also dramatically inhibited thrombosis without disturbing hemostasis.ConclusionThis pharmacological study demonstrated that TYMP participates in multiple signaling pathways in platelet and plays a mechanistic role in regulating platelet activation and thrombosis. TPI, a specific TYMP inhibitor, would be a novel safe anti-platelet and anti-thrombosis medicine.

Using PAR4 Inhibition as an Anti-Thrombotic Approach: Why, How, and When?

Protease-activated receptors (PARs) are a family of four GPCRs with a variety of cellular functions, yet the only advanced clinical endeavours to target these receptors for therapeutic gain to date relates to the impairment of platelet function for anti-thrombotic therapy. The only approved PAR antagonist is the PAR1 inhibitor, vorapaxar—the sole anti-platelet drug against a new target approved in the past 20 years. However, there are two PARs on human platelets, PAR1 and PAR4, and more recent efforts have focused on the development of the first PAR4 antagonists, with first-in-class agents recently beginning clinical trial. Here, we review the rationale for this approach, outline the various modes of PAR4 inhibition, and speculate on the specific therapeutic potential of targeting PAR4 for the prevention of thrombotic conditions.