Effect of Splenectomy on Coagulation and Platelet Function in Adult Liver Transplant Recipients Assessed With Rotational Thromboelastometry and Standard Coagulation Tests.

OBJECTIVES: Splenectomy during liver transplant can affect platelet function. In this study, our primary aim was to assess the perioperative platelet function by rotational thromboelastometry and the effects of splenectomy on platelet function. MATERIALS AND METHODS: We studied 40 consecutive liver transplant recipients with end-stage liver disease (50% as a result of hepatitis C). Patients with splenectomy were compared with patients without splenectomy (n = 20/group). Three platelet function parameters by rotational thromboelastometry were studied: platelet activation with arachidonic acid, platelet activation with adenosine diphosphate, and platelet activation with thrombin receptor-activating peptide 6. Patients were monitored perioperatively and until postoperative day 21. Heparin was infused for 2 days postoperatively (60-180 U/kg/day), followed by administration of subcutaneous low-molecular-weight heparin (40 mg/24 h) on postoperative days 2 and 3 and oral acetylsalicylic acid when platelet count was >50 × 103/µL. RESULTS: Liver disease contributed to low perioperative platelet count and function. Patients showed significant improvement by postoperative day 14 and day 21, particularly after splenectomy. Platelet count was significantly correlated with the 3 platelet function parameters by rotational thromboelastometry (P < .001). Acetyl salicylic acid was required earlier (postoperative day 3) for patients with splenectomy (8/20) but only affected the platelet function represented by platelet activation with arachidonic acid, whereas other platelet activation pathways were less affected. Patients received no transfusions of platelet units. CONCLUSIONS: End-stage liver disease significantly contributed to low platelet function and counts before transplant. Two weeks were required for recovery of patients posttransplant, with further enhancement by splenectomy. Some recipients showed recovery that exceeded the normal reference range, which warranted monitoring. Acetyl salicylic acid only affected 1 platelet activation receptor.

Application of the Cellular Thermal Shift Assay (CETSA) to validate drug target engagement in platelets.

Small molecule drugs play a major role in the study of human platelets. Effective action of a drug requires it to bind to one or more targets within the platelet (target engagement). However, although in vitro assays with isolated proteins can be used to determine drug affinity to these targets, additional factors affect target engagement and its consequences in an intact platelet, including plasma membrane permeability, intracellular metabolism or compartmentalization, and level of target expression. Mechanistic interpretation of the effect of drugs on platelet activity requires comprehensive investigation of drug binding in the proper cellular context, i.e. in intact platelets. The Cellular Thermal Shift Assay (CETSA) is a valuable method to investigate target engagement within complex cellular environments. The assay is based on the principle that drug binding to a target protein increases that protein's thermal stability. In this technical report, we describe the application of CETSA to platelets. We highlight CETSA as a quick and informative technique for confirming the direct binding of drugs to platelet protein targets, providing a platform for understanding the mechanism of action of drugs in platelets, and which will be a valuable tool for investigating platelet signaling and function.

Platelets control blood clotting in health and disease. Small molecule drugs are often used to study human platelets. Here, describe how Cellular Thermal Shift Assay (CETSA) can be used in platelets to investigate the binding between these drugs and their targets inside platelets. This technique can be used to increase our understanding of how existing and future drugs work in platelets.

Translating proof-of-concept for platelet slip into improved antithrombotic therapeutic regimens.

Platelets are central to thrombosis. Research at the intersection of biological and physical sciences provides proof-of-concept for shear rate-dependent platelet slip at vascular stenosis and near device surfaces. Platelet slip extends the observed biological "slip-bonds" to the boundary of functional gliding without contact. As a result, there is diminished engagement of the coagulation cascade by platelets at these surfaces. Comprehending platelet slip would more precisely direct antithrombotic regimens for different shear environments, including for percutaneous coronary intervention (PCI). In this brief report we promote translation of the proof-of-concept for platelet slip into improved antithrombotic regimens by: (1) reviewing new supporting basic biological science and clinical research for platelet slip; (2) hypothesizing the principal variables that affect platelet slip; (3) applying the consequent construct model in support of-and in some cases to challenge-relevant contemporary guidelines and their foundations (including for urgent, higher-risk PCI); and (4) suggesting future research pathways (both basic and clinical). Should future research demonstrate, explain and control platelet slip, then a paradigm shift for choosing and recommending antithrombotic regimens based on predicted shear rate should follow. Improved clinical outcomes with decreased complications accompanying this paradigm shift for higher-risk PCI would also result in substantive cost savings.

1,25-Dihydroxyvitamin D3 attenuates platelet aggregation potentiated by SARS-CoV-2 spike protein via inhibiting integrin αIIbß3 outside-in signaling.

Platelet hyperreactivity contributes to the pathogenesis of COVID-19, which is associated with a hypercoagulability state and thrombosis disorder. It has been demonstrated that Vitamin D deficiency is associated with the severity of COVID-19 infection. Vitamin D supplement is widely used as a dietary supplement due to its safety and health benefits. In this study, we investigated the direct effects and underlying mechanisms of 1,25(OH)2D3 on platelet hyperreactivity induced by SRAS-CoV-2 spike protein via Western blot and platelet functional studies in vitro. Firstly, we found that 1,25(OH)2D3 attenuated platelet aggregation and Src-mediated signaling. We further observed that 1,25(OH)2D3 attenuated spike protein-potentiated platelet aggregation in vitro. Mechanistically, 1,25(OH)2D3 attenuated spike protein upregulated-integrin αIIbß3 outside-in signaling such as platelet spreading and the phosphorylation of ß3, c-Src and Syk. Moreover, using PP2, the Src family kinase inhibitor to abolish spike protein-stimulated platelet aggregation and integrin αIIbß3 outside-in signaling, the combination of PP2 and 1,25(OH)2D3 did not show additive inhibitory effects on spike protein-potentiated platelet aggregation and the phosphorylation of ß3, c-Src and Syk. Thus, our data suggest that 1,25(OH)2D3 attenuates platelet aggregation potentiated by spike protein via downregulating integrin αIIbß3 outside-in signaling.

Off-target effect of high-dose sildenafil on adenosine 5'- diphosphate and collagen-induced platelet activation through mitogen-activated protein kinase pathway in treated BALB/C mice and in vitro experiments: A preliminary study.

ABSTRACT: Sildenafil, a common over-the-counter pill often self-administered at high doses for erectile dysfunction, has been reported to rarely cause prothrombotic events and sudden cardiac death in a few case reports. Therefore, we investigated the in vitro and in vivo effect of sildenafil treatment and dosage on platelet activation and mitogen-activated protein kinase (MAPK) phosphorylation. BALB/C mice were segregated into four groups, each having four mice each (control, low [3.25 mg/kg], medium [6.5 mg/kg], and high [13 mg/kg] sildenafil), and after the treatment, blood was drawn from each mouse and washed platelets prepared. Washed platelets were incubated with CD41 PE-Cy7 and Phospho-p38 MAPK PE antibodies and analyzed using a flow cytometer for platelet activation and adenosine 5'- diphosphate (ADP)/collagen-induced MAPK phosphorylation. Washed platelets obtained from the venous blood of 18 human volunteers, were incubated with PAC-1 FITC and Phospho-p38 MAPK PE antibodies, and platelet activation (ADP and collagen), followed by flow cytometry analysis. There was a significant increase in both platelet activation as well as MAPK phosphorylation in the presence of collagen in the high-dose (13 mg/kg) sildenafil group (P = 0.000774). Further, increased platelet activation was observed in samples that were treated with high-dose sildenafil as compared to the untreated samples (P < 0.00001). These studies show the risk of prothrombotic episodes in patients on high-dose sildenafil (100 mg), in those with even mild endothelial dysfunction due to ADP, and collagen-induced platelet activation through MAPK phosphorylation, which was not seen in the low-and intermediate-dose cohorts.

C-type lectin-like receptor-2 in platelets mediates ferric chloride-induced platelet activation and attenuates ferroptosis of endothelial cells.

BACKGROUND: An iron overload status induces ferroptosis, an iron-dependent nonapoptotic cell death, in various pathological conditions. We previously reported that hemin (heme), protoporphyrin-IX with ferric iron, activates platelets via C-type lectin-like receptor-2 (CLEC-2) and glycoprotein VI/FcRγ, but protoporphyrin-IX alone blocks CLEC-2-dependent platelet activation. Therefore, we hypothesized that free iron has the ability to activate platelets. OBJECTIVES: This study aimed to elucidate platelet activation mechanisms of iron (ferric chloride), including the identification of signaling pathways and receptors, and to examine whether platelets regulate ferroptosis. METHODS: Platelet aggregometry, platelet activation marker expression, and protein phosphorylation were examined in ferric chloride-stimulated human and murine platelets. Inhibitors of platelet activation signaling pathways and receptor-deleted platelets were utilized to identify the responsible signaling pathway and receptor. The effect of platelets on ferroptosis of endothelial cells was investigated in vitro. RESULTS: Ferric chloride induced platelet activation dependent on Src family kinase pathways in humans and mice. Ferric chloride-induced platelet aggregation was almost lost in CLEC-2-depleted murine platelets and wild-type platelets preincubated with recombinant CLEC-2 proteins. Furthermore, coculture of wild-type platelets, but not CLEC-2-deficient platelets, attenuated ferroptosis of endothelial cells in vitro. CONCLUSION: Ferric chloride activates platelets via CLEC-2 and Src family kinase pathways, and platelets have a protective role in the ferroptosis of endothelial cells dependent on CLEC-2.

Platelets favor the outgrowth of established metastases.

Despite abundant evidence demonstrating that platelets foster metastasis, anti-platelet agents have low therapeutic potential due to the risk of hemorrhages. In addition, whether platelets can regulate metastasis at the late stages of the disease remains unknown. In this study, we subject syngeneic models of metastasis to various thrombocytopenic regimes to show that platelets provide a biphasic contribution to metastasis. While potent intravascular binding of platelets to tumor cells efficiently promotes metastasis, platelets further support the outgrowth of established metastases via immune suppression. Genetic depletion and pharmacological targeting of the glycoprotein VI (GPVI) platelet-specific receptor in humanized mouse models efficiently reduce the growth of established metastases, independently of active platelet binding to tumor cells in the bloodstream. Our study demonstrates therapeutic efficacy when targeting animals bearing growing metastases. It further identifies GPVI as a molecular target whose inhibition can impair metastasis without inducing collateral hemostatic perturbations.

Effect of plateletcrit and methylenetetrahydrofolate reductase (MTHFR) C677T genotypes on folic acid efficacy in stroke prevention.

Previous studies have shown that low platelet count combined with high plasma total homocysteine (tHcy) increased stroke risk and can be lowered by 73% with folic acid. However, the combined role of other platelet activation parameters and the methylenetetrahydrofolate reductase (MTHFR) C677T genotypes on stroke risk and folic acid treatment benefit remain to be examined. This study aimed to investigate if platelet activation parameters and MTHFR genotypes jointly impact folic acid treatment efficacy in first stroke prevention. Data were derived from the China Stroke Primary Prevention Trial. This study includes a total of 11,185 adult hypertensive patients with relevant platelet activation parameters and MTHFR genotype data. When simultaneously considering both platelet activation parameters (plateletcrit, platelet count, mean platelet volume, platelet distribution width) and MTHFR genotypes, patients with both low plateletcrit (Q1) and the TT genotype had the highest stroke incidence rate (5.6%) in the enalapril group. This subgroup significantly benefited from folic acid treatment, with a 66% reduction in first stroke (HR: 0.34; 95% CI: 0.14-0.82; p = 0.016). Consistently, the subgroup with low plateletcrit (Q1) and the CC/CT genotype also benefited from folic acid treatment (HR: 0.40; 95% CI: 0.23-0.70; p = 0.001). In Chinese hypertensive adults, low plateletcrit can identify those who may greatly benefit from folic acid treatment, in particular, those with the TT genotype, a subpopulation known to have the highest stroke risk.

Antiplatelet Effects of Flavonoid Aglycones Are Mediated by Activation of Cyclic Nucleotide-Dependent Protein Kinases.

Flavonoid aglycones are secondary plant metabolites that exhibit a broad spectrum of pharmacological activities, including anti-inflammatory, antioxidant, anticancer, and antiplatelet effects. However, the precise molecular mechanisms underlying their inhibitory effect on platelet activation remain poorly understood. In this study, we applied flow cytometry to analyze the effects of six flavonoid aglycones (luteolin, myricetin, quercetin, eriodictyol, kaempferol, and apigenin) on platelet activation, phosphatidylserine externalization, formation of reactive oxygen species, and intracellular esterase activity. We found that these compounds significantly inhibit thrombin-induced platelet activation and decrease formation of reactive oxygen species in activated platelets. The tested aglycones did not affect platelet viability, apoptosis induction, or procoagulant platelet formation. Notably, luteolin, myricetin, quercetin, and apigenin increased thrombin-induced thromboxane synthase activity, which was analyzed by a spectrofluorimetric method. Our results obtained from Western blot analysis and liquid chromatography-tandem mass spectrometry demonstrated that the antiplatelet properties of the studied phytochemicals are mediated by activation of cyclic nucleotide-dependent signaling pathways. Specifically, we established by using Förster resonance energy transfer that the molecular mechanisms are, at least partly, associated with the inhibition of phosphodiesterases 2 and/or 5. These findings underscore the therapeutic potential of flavonoid aglycones for clinical application as antiplatelet agents.

Zixue Powder attenuates septic thrombosis via reducing neutrophil extracellular trap through blocking platelet STING activation.

ETHNOPHARMACOLOGICAL RELEVANCE: Microthrombosis is commonly seen in sepsis and COVID-19. Zixue Powder (ZXP) is a traditional Chinese herbal formula with the potential to treat microvascular and infectious diseases. However, the role and mechanism of ZXP in sepsis-associated thrombosis remain unclear. AIM OF THE STUDY: Investigating the therapeutic effectiveness and underlying mechanisms of ZXP in septic thrombosis. MATERIALS AND METHODS: ZXP's compositions were examined with UPLC-QTOF-MS. The efficacy of ZXP on sepsis-induced thrombosis was assessed through various methods: liver tissue pathology was examined using hematoxylin-eosin staining, platelet count was determined by a blood cell analyzer, and an enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of serum tissue factor (TF), thromboxane B2 (TXB2), D-Dimer, and plasminogen activator inhibitor-1 (PAI-1). Neutrophil extracellular traps (NETs) were localized and expressed in liver tissues by immunofluorescence, and the number of NETs in peripheral blood was evaluated by ELISA, which measured the quantity of cf-DNA and MPO-DNA in serum. Platelet P-selectin expression and platelet-neutrophil aggregation were measured by flow cytometry, and plasma P-selectin expression was measured by ELISA. Furthermore, the mechanism of the stimulator of interferon genes (STING) signaling pathway in ZXP's anti-sepsis thrombosis effect was investigated using the STING agonist, Western blot experiments, and immunoprecipitation experiments. RESULTS: UPLC-QTOF-MS identified 40 chemical compositions of ZXP. Administration of ZXP resulted in significant improvements in liver thrombosis, platelet counts, and levels of TXB2, TF, PAI-1, and D-Dimer in septic rats. Moreover, ZXP inhibited NETs formation in both liver tissue and peripheral blood. Additionally, ZXP decreased the levels of P-selectin in both platelets and plasma, as well as the formation of platelet-neutrophil aggregates, thereby suppressing P-selectin-mediated NETs release. Immunoprecipitation and immunofluorescence staining experiments revealed that ZXP attenuated P-selectin secretion by inhibiting STING-mediated assembly of platelet soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) complex, ultimately preventing inhibition of NETs formation. CONCLUSION: Our study showed that ZXP effectively mitigates platelet granule secretion primarily through modulation of the STING pathway, consequently impeding NET-associated thrombosis in sepsis. These findings offer valuable insights for future research on the development and application of ZXP.

Safety, Tolerability, and Pharmacodynamics of AZD3366 (Optimized Human CD39L3 Apyrase) Alone and in Combination With Ticagrelor and Acetylsalicylic Acid: A Phase 1, Randomized, Placebo-Controlled Study.

BACKGROUND: ADP and ATP are importantly involved in vascular and thrombotic homeostasis, via multiple receptor pathways. Blockade of ADP P2Y12 receptors inhibits platelet aggregation and represents an effective cardiovascular disease prevention strategy. AZD3366 (APT102), a long-acting recombinant form of an optimized CD39L3 human apyrase, has effectively reduced ATP, ADP, and platelet aggregation and provided tissue protection in preclinical models, features that could be very beneficial in treating patients with cardiovascular disease. METHODS AND RESULTS: We conducted this phase 1, first-in-human study of single ascending doses of intravenous AZD3366 or placebo, including doses added to dual antiplatelet therapy with ticagrelor and acetylsalicylic acid. The primary objective was safety and tolerability; secondary and exploratory objectives included pharmacokinetics, pharmacodynamics (measured as inhibition of platelet aggregation), adenosine diphosphatase (ADPase) activity, and ATP/ADP metabolism. In total, 104 participants were randomized. AZD3366 was generally well tolerated, with no major safety concerns observed. ADPase activity increased in a dose-dependent manner with a strong correlation to AZD3366 exposure. Inhibition of ADP-stimulated platelet aggregation was immediate, substantial, and durable. In addition, there was a prompt decrease in systemic ATP concentration and an increase in adenosine monophosphate concentrations, whereas ADP concentration appeared generally unaltered. At higher doses, there was a prolongation of capillary bleeding time without detectable changes in the ex vivo thromboelastometric parameters. CONCLUSIONS: AZD3366 was well tolerated in healthy participants and demonstrated substantial and durable inhibition of platelet aggregation after single dosing. Higher doses prolonged capillary bleeding time without detectable changes in ex vivo thromboelastometric parameters. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique Identifier: NCT04588727.

Assessment of platelet functional activity in healthy individuals and patients receiving antiplatelet therapy. Possible inconsistencies between aggregation and flow cytometry tests.

Platelet functional activity was assessed in healthy volunteers (HV, n=92), patients with stable angina pectoris (SA, n=42) and acute coronary syndrome (ACS, n=73), treated with acetylsalicylic acid (ASA) + clopidogrel and ASA + ticagrelor, respectively. In all HV and patients we have compared parameters of platelet aggregation (maximum light transmission and velocity, Tmax and Vmax) and parameters, characterizing exposure of platelet activation markers, evaluated by flow cytometry. HV platelets were activated by 10 µM, 1 µM TRAP, and 20 µM, 5 µM, 2.5 µM ADP; patient platelets were activated by 10 µM TRAP and by 20 µM and 5 µM ADP. Strong and significant correlations between the aggregation and flow cytometry parameters (the r correlation coefficient from 0.4 up to >0.6) most frequently were registered in HV platelet during activation by 1 µM TRAP and in SA patients during platelet activation by 20 µM and 5 µM ADP. However, in many other cases these correlations were rather weak (r < 0.3) and sometimes statistically insignificant. In HV the differences in PAC-1 binding parameters between platelets activated by 10 µM TRAP (the strongest agonist) and all ADP concentrations were negligible (≤ 10%), while CD62P binding (at all ADP concentrations) and LTA parameters for (5 µM and 2.5 µM ADP) were significantly lower (by 40-60%). Antiplatelet therapy in patients decreased all parameters as compared to HV, but to varying extents. For 10 µM TRAP the MFI index for PAC-1 binding (40-50% decrease) and for both ADP concentrations the Tmax values (60-85% decrease) appeared to be the most sensitive in comparison with the other parameters that decreased to a lesser extent. The data obtained indicate a possibility of inconsistency between different LTA and flow cytometry parameters in assessing platelet activity and efficacy of antiplatelet drugs.

Investigating the role of rotenone on human blood platelets: Molecular insights into abnormal platelet functions in Parkinson's disease.

Parkinson's disease (PD) is a predominant neuromotor disorder characterized by the selective death of dopaminergic neurons in the midbrain. The majority of PD cases are sporadic or idiopathic, with environmental toxins and pollutants potentially contributing to its development or exacerbation. However, clinical PD patients are often associated with a reduced stroke frequency, where circulating blood platelets are indispensable. Although platelet structural impairment is evident in PD, the platelet functional alterations and their underlying molecular mechanisms are still obscure. Therefore, we investigated rotenone (ROT), an environmental neurotoxin that selectively destroys dopaminergic neurons mimicking PD, on human blood platelets to explore its impact on platelet functions, thus replicating PD conditions in vitro. Our study deciphered that ROT decreased thrombin-induced platelet functions, including adhesion, activation, secretion, and aggregation in human blood platelets. As ROT is primarily responsible for generating intracellular reactive oxygen species (ROS), and ROS is a key player regulating the platelet functional parameters, we went on to check the effect of ROT on platelet ROS production. In our investigation, it became evident that ROT treatment resulted in the stimulation of ROS production in human blood platelets. Additionally, we discovered that ROT induced ROS production by augmenting Ca2+ mobilization from inositol 1,4,5-trisphosphate receptor. Apart from this, the treatment of ROT triggers protein kinase C associated NADPH oxidase-mediated ROS production in platelets. In summary, this research, for the first time, highlights ROT-induced abnormal platelet functions and may provide a mechanistic insight into the altered platelet activities observed in PD patients.

Cisplatin-loaded mesoporous polydopamine nanoparticles capped with MnO2 and coated with platelet membrane provide synergistic anti-tumor therapy.

A multifunctional nanoplatform was constructed in this work, with the goal of ameliorating the challenges faced with traditional cancer chemotherapy. Cisplatin (CP) was loaded into mesoporous polydopamine (mPDA) nanoparticles (NPs) with a drug loading of 15.8 ± 0.1 %, and MnO2 used as pore sealing agent. Finally, the NPs were wrapped with platelet membrane (PLTM). P-selectin on the PLTM can bind to CD44, which is highly expressed on the tumor cell membrane, so as to improve the targeting performance of the NPs. In addition, the CD47 on the PLTM can prevent the NPs from being phagocytosed by macrophages, which is conducive to immune escape. The final PLTM-CP@mPDA/MnO2 NPs were found to have a particle size of approximately 198 nm. MnO2 is degraded into Mn2+ in the tumor microenvironment, leading to CP release from the pores in the mPDA. CP both acts as a chemotherapy agent and can also increase the concentration of H2O2 in cells. Mn2+ can catalyze the conversion of H2O2 to OH, resulting in oxidative damage and chemodynamic therapy. In addition, Mn2+ can be used as a contrast agent in magnetic resonance imaging (MRI). In vitro and in vivo experiments were performed to explore the therapeutic effect of the NPs. When the concentration of CP is 30 µg/mL, the NPs cause approximately 50 % cell death. It was found that the PLTM-CP@mPDA/MnO2 NPs are targeted to cancerous cells, and in the tumor site cause extensive apoptosis. Tumor growth is thereby repressed. No negative off-target side effects were noted. MRI could be used to confirm the presence of the NPs in the tumor site. Overall, the nano-platform developed here provides cooperative chemotherapy and chemodynamic therapy, and can potentially be used for effective cancer treatment which could be monitored by MRI.

Oxidative Stress-Induced Platelet Apoptosis/Activation: Alleviation by Purified Curcumin via ASK1-JNK/p-38 Pathway.

Platelets are known for their indispensable role in hemostasis and thrombosis. However, alteration in platelet function due to oxidative stress is known to mediate various health complications, including cardiovascular diseases and other health complications. To date, several synthetic molecules have displayed antiplatelet activity; however, their uses are associated with bleeding and other adverse effects. The commercially available curcumin is generally a mixture of three curcuminoids: curcumin, demethoxycurcumin, and bisdemethoxycurcumin. Although crude curcumin is known to inhibit platelet aggregation, the effect of purified curcumin on platelet apoptosis, activation, and aggregation remains unclear. Therefore, in this study, curcumin was purified from a crude curcumin mixture and the effects of this preparation on the oxidative stress-induced platelet apoptosis and activation was evaluated. 2,2'-Azobis(2-methylpropionamidine) dihydrochloride (AAPH) compound was used as an inducer of oxidative stress. Purified curcumin restored AAPH-induced platelet apoptotic markers like reactive oxygen species, intracellular calcium level, mitochondrial membrane potential, cardiolipin peroxidation, cytochrome c release from mitochondria to the cytosol, and phosphatidyl serine externalization. Further, it inhibited the agonist-induced platelet activation and aggregation, demonstrating its antiplatelet activity. Western blot analysis confirms protective effect of the purified curcumin against oxidative stress-induced platelet apoptosis and activation via downregulation of MAPKs protein activation, including ASK1, JNK, and p-38. Together, these results suggest that the purified curcumin could be a potential therapeutic bioactive molecule to treat the oxidative stress-induced platelet activation, apoptosis, and associated complications.

Genetically encoded biocompatible anti-coagulant protein-coated coronary artery stents drive endothelialization.

In response to the critical demand for advancements in coronary artery stents, this study addresses the challenges associated with arterial recoil and restenosis post-angioplasty and the imperative to encourage rapid re-endothelialization for minimizing thrombosis risks. We employed an innovative approach inspired by mussel adhesion, incorporating placental anticoagulant protein (AnnexinV) on stent design. The introduction of a post-translationally modified catecholic amino acid L-3,4-dihydroxyphenylalanine (L-Dopa), mimicking mussel characteristics, allowed for effective surface modification of Stainless steel stents through genetic code engineering in AnnexinV (AnxDopa). The efficacy of AnxDopa was analyzed through microscale thermophoresis and flow cytometry, confirming AnxDopa's exceptional binding with phosphatidylserine and activated platelets. AnxDopa coated stainless steel demonstrates remarkable bio-, hemo-, and immuno-compatibility, preventing smooth muscle cell proliferation, platelet adhesion, and fibrin formation. It acts as an interface between the stent and biological fluid, which facilitates the anticoagulation and rapid endothelialization. Surface modification of SS verified through XPS analysis and contact angle measurement attests to the efficacy of AnxDopa mediated surface modification. The hydrophilic nature of the AnxDopa-coated surface enhanced the endothelialization through increased protein absorption. This approach represents a significant stride in developing coronary stents with improved biocompatibility and reduced restenosis risks, offering valuable contributions to scientific and clinical realms alike.

Individualized antiplatelet therapy for non-cardiogenic ischemic stroke.

OBJECTIVE: This research aims to investigate the impact of individualized antiplatelet therapy guided by thromboelastography with platelet mapping (TEG-PM) on the clinical outcomes of patients with non-cardiogenic ischemic stroke. METHODS: Among a total of 1264 patients, 684 individuals diagnosed with non-cardiogenic ischemic stroke underwent TEG-PM testing. Based on the adjustment of antiplatelet medication, these patients were divided into individual and control groups. Within the individual group, in accordance with the TEG-PM test results, a Maximum amplitude (MA) value greater than 47mm was defined as high residual platelet reactivity (HRPR), while an MA value less than 31mm was defined as low residual platelet reactivity (LRPR). Patients with arachidonic acid (AA) less than 50% and adenosine diphosphate (ADP) less than 30% were classified as aspirin-resistant or clopidogrel-resistant. Treatment strategies for antiplatelet medication were subsequently adjusted accordingly, encompassing increment, decrement, or replacement of drugs. Meanwhile, the control group maintained their original medication regimen without alterations. RESULTS: The individual group included 487 patients, while the control group had 197. In the individual group, approximately 175 patients (35.9%) were treated with increased medication dosages, 89 patients (18.3%) with reduced dosages, and 223 patients (45.8%) switched medications. The results showed that the incidence rate of ischemic events in the individual group was lower than that of the control group (5.54% vs. 12.6%, P = 0.001), but no significant difference was observed in bleeding events. Cox regression analysis revealed age (hazard ratio, 1.043; 95% CI, 1.01-1.078; P = 0.011) and coronary heart disease (hazard ratio, 1.902; 95% CI, 1.147-3.153; P = 0.013) as significant risk factors for adverse events. CONCLUSION: Individualized antiplatelet therapy based on TEG-PM results can reduce the risk of ischemic events in patients with non-cardiogenic ischemic stroke without increasing the risk of bleeding events or mortality. Advanced age and coronary heart disease were identified as risk factors affecting the outcomes of individualized antiplatelet therapy.

Qilong capsule prevents myocardial ischemia/reperfusion injury by inhibiting platelet activation via the platelet CD36 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Qilong capsule (QC) is developed from the traditional Chinese medicine formula Buyang Huanwu Decoction, which has been clinically used to invigorate Qi and promote blood circulation to eliminate blood stasis. Myocardial ischemia‒reperfusion injury (MIRI) can be attributed to Qi deficiency and blood stasis. However, the effects of QC on MIRI remain unclear. AIM OF THE STUDY: This study aimed to investigate the protective effect and possible mechanism of QC on platelet function in MIRI rats. MATERIALS AND METHODS: The left anterior descending artery of adult Sprague‒Dawley rats was ligated for 30 min and then reperfused for 120 min with or without QC treatment. Then, the whole blood viscosity, plasma viscosity, coagulation, platelet adhesion rate, platelet aggregation, and platelet release factors were evaluated. Platelet CD36 and its downstream signaling pathway-related proteins were detected by western blotting. Furthermore, the active components of QC and the molecular mechanism by which QC regulates platelet function were assessed via molecular docking, platelet aggregation tests in vitro and BLI analysis. RESULTS: We found that QC significantly reduced the whole blood viscosity, plasma viscosity, platelet adhesion rate, and platelet aggregation induced by ADP or AA in rats with MIRI. The inhibition of platelet activation by QC was associated with reduced levels of ß-TG, PF-4, P-selectin and PAF. Mechanistically, QC effectively attenuated the expression of platelet CD36 and thus inhibited the activation of Src, ERK5, and p38. The active components of QC apparently suppressed platelet aggregation in vitro and regulated the CD36 signaling pathway. CONCLUSIONS: QC improves MIRI-induced hemorheological disorders, which might be partly attributed to the inhibition of platelet activation via CD36-mediated platelet signaling pathways.

Platelet Membrane-Coated r-SAK Improves Thrombolytic Efficacy by Targeting Thrombus.

Thrombolytic therapy is one of the most effective treatments for thrombus dissolution and recanalization of blocked vessels in thrombotic diseases. However, the application of the thrombolytic strategy has been limited due to unsatisfactory thrombolytic efficacy, relatively higher bleeding complications, and consequently restricted indications. Recombinant staphylokinase (r-SAK) is a third-generation thrombolytic agent produced by genetic engineering technology, which exhibits a better thrombolytic efficacy than urokinase and recombinant streptokinase. Inspired by the natural affinity of platelets in hemostasis and pathological thrombosis, we developed a platelet membrane (PM)-coated r-SAK (PM-r-SAK). Results from animal experiments and human in vitro studies showed that the PM-r-SAK had a thrombolytic efficacy equal to or better than its 4-fold dose of r-SAK. In a totally occluded rabbit femoral artery thrombosis model, the PM-r-SAK significantly shortened the initial recanalization time compared to the same dose and 4-fold dose of r-SAK. Regarding the recanalized vessels, the PM-r-SAK prolonged the time of reperfusion compared to the same dose and 4-fold dose of r-SAK, though the differences were not significant. An in vitro thrombolytic experiment demonstrated that the thrombolytic efficacy of PM-r-SAK could be inhibited by platelet-poor plasma from patients taking aspirin and ticagrelor. PM coating significantly improves the thrombolytic efficacy of r-SAK, which is related to the thrombus-targeting activity of the PM-r-SAK and can be inhibited by aspirin- and ticagrelor-treated plasma.