The Combination of Natural Compounds Escin-Bromelain-Ginkgo Biloba-Sage Miltiorrhiza (EBGS) Reduces Platelet Adhesion to TNFα-Activated Vascular Endothelium through FAK Signaling.

Thrombosis is a key process that determines acute coronary syndrome and ischemic stroke and is the leading cause of morbidity and mortality in the world, together with cancer. Platelet adhesion and subsequent activation and aggregation are critical processes that cause thrombus formation after endothelial damage. To date, high hopes are associated with compounds of natural origin, which show anticoagulant action without undesirable effects and can be proposed as supportive therapies. We investigated the effect of the new combination of four natural compounds, escin-bromelain-ginkgo biloba-sage miltiorrhiza (EBGS), on the initial process of the coagulation cascade, which is the adhesion of platelets to activated vascular endothelium. Our results demonstrated that EBGS pretreatment of endothelial cells reduces platelet adhesion even in the presence of the monocyte-lymphocyte population. Our data indicate that EBGS exerts its effects by inhibiting the transcription of adhesion molecules, including P-selectin, platelet membrane glycoprotein GP1b, integrins αV and ß3, and reducing the secretion of the pro-inflammatory cytokines interleukin 6, interleukin 8, and the metalloproteinases MMP-2 and MMP-9. Furthermore, we demonstrated that EBGS inhibited the expression of focal adhesion kinase (FAK), strictly involved in platelet adhesion, and whose activity is correlated with that of integrin ß3. The results shown in this manuscript suggest a possible inhibitory role of the new combination EBGS in the reduction in platelet adhesion to activated endothelium, thus possibly preventing coagulation cascade initiation.

Platelet Biorheology and Mechanobiology in Thrombosis and Hemostasis: Perspectives from Multiscale Computation.

Thrombosis is the pathological clot formation under abnormal hemodynamic conditions, which can result in vascular obstruction, causing ischemic strokes and myocardial infarction. Thrombus growth under moderate to low shear (<1000 s-1) relies on platelet activation and coagulation. Thrombosis at elevated high shear rates (>10,000 s-1) is predominantly driven by unactivated platelet binding and aggregating mediated by von Willebrand factor (VWF), while platelet activation and coagulation are secondary in supporting and reinforcing the thrombus. Given the molecular and cellular level information it can access, multiscale computational modeling informed by biology can provide new pathophysiological mechanisms that are otherwise not accessible experimentally, holding promise for novel first-principle-based therapeutics. In this review, we summarize the key aspects of platelet biorheology and mechanobiology, focusing on the molecular and cellular scale events and how they build up to thrombosis through platelet adhesion and aggregation in the presence or absence of platelet activation. In particular, we highlight recent advancements in multiscale modeling of platelet biorheology and mechanobiology and how they can lead to the better prediction and quantification of thrombus formation, exemplifying the exciting paradigm of digital medicine.

Angiogenesis outcomes of metformin utilization in diabetes mellitus: a systematic review and meta-analysis.

OBJECTIVE: To review relevant literature regarding the role of metformin in angiogenesis among diabetic patients. METHODS: The systematic review and meta-analysis conducted from May to September 2022, and comprised search on Medline, ScienceDirect, ProQuest, Web of Science, EBSCOhost and Cochrane Library databases. The studies included were published in the English language and were human studies having angiogenesis endothelial markers as the outcomes of interest among patients of type 2 diabetes mellitus undergoing metformin therapy. Endothelial markers, including vascular endothelial growth factor, von-Willebrand-factor, plasminogen activator inhibitor-1, soluble vascular adhesion molecule- 1, intercellular adhesion molecule-1, soluble endothelialselectin, tissue plasminogen activator, urinary albumin excretion, platelet endothelial cell adhesion molecule-1 and thrombin-activatable fibrinolysis inhibitor, were assessed as angiogenesis outcomes. Data was statistically analysed using Review Manager 5.4. RESULTS: Of the 413 studies identified, 8(1.9%) were included; 5(62.5%) randomised control trials, 2(25.0%) cross-sectional, and 1(12.5%) cohort studies, with overall 1199 patients. Among the outcomes, von-Willebrandfactor (p=0.01), soluble vascular adhesion molecule-1 (p<0.00001), intercellular adhesion molecule-1 (p=0.0003), soluble endothelial-selectin (p=0.007), and tissue plasminogen activator (p<0.00001) showed significantly lower levels after metformin treatment using the random effect methods. CONCLUSIONS: Metformin was found to have an additional effect of endothelial function improvement.

Sodium bicarbonate as a local adjunctive agent for limiting platelet activation, aggregation, and adhesion within cardiovascular therapeutic devices.

Cardiovascular therapeutic devices (CTDs) remain limited by thrombotic adverse events. Current antithrombotic agents limit thrombosis partially, often adding to bleeding. The Impella® blood pump utilizes heparin in 5% dextrose (D5W) as an internal purge to limit thrombosis. While effective, exogenous heparin often complicates overall anticoagulation management, increasing bleeding tendency. Recent clinical studies suggest sodium bicarbonate (bicarb) may be an effective alternative to heparin for local anti-thrombosis. We examined the effect of sodium bicarbonate on human platelet morphology and function to better understand its translational utility. Human platelets were incubated (60:40) with D5W + 25 mEq/L, 50 mEq/L, or 100 mEq/L sodium bicarbonate versus D5W or D5W + Heparin 50 U/mL as controls. pH of platelet-bicarbonate solutions mixtures was measured. Platelet morphology was examined via transmission electron microscopy; activation assessed via P-selectin expression, phosphatidylserine exposure and thrombin generation; and aggregation with TRAP-6, calcium ionophore, ADP and collagen quantified; adhesion to glass measured via fluorescence microscopy. Sodium bicarbonate did not alter platelet morphology but did significantly inhibit activation, aggregation, and adhesion. Phosphatidylserine exposure and thrombin generation were both reduced in a concentration-dependent manner-between 26.6 ± 8.2% (p = 0.01) and 70.7 ± 5.6% (p < 0.0001); and 14.0 ± 6.2% (p = 0.15) and 41.7 ± 6.8% (p = 0.03), respectively, compared to D5W control. Platelet aggregation via all agonists was also reduced, particularly at higher concentrations of bicarb. Platelet adhesion to glass was similarly reduced, between 0.04 ± 0.03% (p = 0.61) and 0.11 ± 0.04% (p = 0.05). Sodium bicarbonate has direct, local, dose-dependent effects limiting platelet activation and adhesion. Our results highlight the potential utility of sodium bicarbonate as a locally acting agent to limit device thrombosis.

Filamin A regulates caspase-3 cleavage in platelets in a protein kinase C (PKC)-dependent manner.

Apoptosis is a critical process for the maintenance of cell populations, and involves mitochondrial depolarization, the sequential cleavage of caspase-9 and -3, followed by the externalization of phosphatidylserine (PS) on the plasma membrane. The actin cytoskeleton and its accessory proteins are known regulators of apoptotic signaling in nucleated cells but their roles in platelet apoptosis are undefined. Filamin A (FLNA) is a ubiquitously expressed actin-crosslinking protein that also serves as an intracellular signaling scaffold. Here we used platelets from mice with a platelet-specific FLNA deficiency (Flnafl/Y, Pf4-cre/+, termed platelet-specific knockout) to test the role of FLNA in platelet apoptosis. Treatment with the BH3-mimetic drug ABT-737 induced caspase-3 cleavage and PS exposure in platelets from floxed mice (Flnafl/Y, termed control) but these effects were essentially abrogated in FLNA-null platelets (platelet-specific knockout). Protein kinase C (PKC), a known FLNA ligand, was also activated by ABT-737, and PKC's phosphorylation of its downstream substrates was attenuated in FLNA-null platelets. The PKC inhibitor bisindolylmaleimide (BIM) also reduced caspase-3 cleavage, thus essentially phenocopying the FLNA-null platelets. Notably, the caspase-3 cleavage defect in FLNA-null platelets was rescued by the PKC-activating phorbol ester PMA, suggesting that FLNA and PKC share a common pathway in regulating platelet apoptosis. Mitochondrial depolarization and caspase-9 cleavage were unaffected by BIM treatment, suggesting that PKC specifically controls the downstream caspase-3 point of the pro-apoptotic signaling pathway. These data point to a novel role for FLNA in the regulation of platelet apoptosis, thus providing an improved understanding of how circulating platelet counts are maintained.

α1-Adrenergic Stimulation Increases Platelet Adhesion to Endothelial Cells Mediated by TRPC6.

Stimulation of a1-adrenergic nervous system is increased during systemic inflammation and other pathological conditions with the consequent adrenergic receptors (ARs) activation. It has been reported that a1-stimulation contributes to coagulation since a1-AR blockers inhibit coagulation and its organic consequences. Also, coagulation induced by a1-AR stimulation can be greatly decreased using a1-AR blockers. In health, endothelial cells (ECs) perform anticoagulant actions at cellular and molecular level. However, during inflammation, ECs turn dysfunctional promoting a procoagulant state. Endothelium-dependent coagulation progresses at cellular and molecular levels, promoting endothelial acquisition of procoagulant properties to potentiate coagulation by means of prothrombotic and antifibrinolytic proteins expression increase in ECs releasing them to circulation, the thrombus formation is strengthened. Calcium signaling is a main feature of coagulation. Inhibition of ion channels involved in Ca2+ entry severely decreases coagulation. The transient receptor potential canonical 6 (TRPC6) is a non-selective Ca2+-permeable ion channel. TRPC6 activity is induced by diacylglycerol, suggesting that is regulated by a1-ARs. Furthermore, a1-ARs stimulation elicits a TRPC-like current in rat mesenteric artery smooth muscle and mesangial cells. However, whether TRPC6 could promote an ECs-mediated platelet adhesion induced by a1-adrenergic stimulation is currently not known. Therefore, the aim of this study was to examine if the TRPC6 calcium channel mediates platelet adhesion induced by a1-adrenergic stimulation. Our results suggest that platelet adhesion to ECs is enhanced by the a1-adrenergic stimulation evoked by phenylephrine mediated by TRPC6 activity. We conclude that TRPC6 is a molecular determinant in platelet adhesion to ECs with implications in systemic inflammatory diseases treatment.

SARS-CoV-2 Spike Protein and Neutralizing Anti-Spike Protein Antibodies Modulate Blood Platelet Function.

Several studies report elevated blood platelet activation and altered platelet count in COVID-19 patients, but the role of the SARS-CoV-2 spike protein in this process remains intriguing. Additionally, there is no data that anti-SARS-CoV-2 neutralizing antibodies (nAb) may attenuate spike protein activity toward blood platelets. Our results indicate that under in vitro conditions, the spike protein increased the collagen-stimulated aggregation of isolated platelets and induced the binding of vWF to platelets in ristocetin-treated blood. The spike protein also significantly reduced collagen- or ADP-induced aggregation or decreased GPIIbIIIa (fibrinogen receptor) activation in whole blood, depending on the presence of the anti-spike protein nAb. Our findings suggest that studies on platelet activation/reactivity in COVID-19 patients or in donors vaccinated with anti-SARS-CoV-2 and/or previously-infected COVID-19 should be supported by measurements of spike protein and IgG anti-spike protein antibody concentrations in blood.

SdrG gene activated joint surface membrane protein PTPRJ to induce periprosthetic joint infection after total hip arthroplasty.

WHAT IS KNOWN AND OBJECTIVE: Up to now, no study focused on the role of SdrG in PJI after THA. To explore the mechanism of periprosthetic joint infection (PJI) after total hip arthroplasty (THA). METHODS: Joint fluid and blood were collected from patients with PJI after THA, aseptic loosening of the joints or bacterial infection after traumatic fractures of the extremities alone. The expression of SdrG in the 3 groups was determined by agarose gel electrophoresis. The expression of protein tyrosine phosphatase receptor J (PTPRJ) was measured by immunohistochemistry method. The platelet adhesion rate was determined by biochemical analysis. The content of D-dimer, CRP and ESR in blood was determined by latex agglutination method. Multiple linear correlation analysis was used to analyse the correlation between PJI and the expression of PTPRJ protein, platelet adhesion rate, D-dimer content, CRP and ESR. RESULTS AND DISCUSSION: The expression of SdrG and PTPRJ in PJI group was markedly increased compared to the other 2 groups. The platelet adhesion rate in PJI group was markedly larger compared to aseptic loosening and simple wound infection group, and the rate in simple wound infection group was larger than aseptic loosening group. The level of D-dimer, CRP and ESR in PJI group was higher than that of the other groups. The expression of PTPRJ protein, D-dimer content, CRP and ESR was all closely related to PJI, while platelet adhesion rate had no correlation with PJI. WHAT IS NEW AND CONCLUSION: SDRG gene around joint prosthesis was over-expressed, which activated joint surface membrane protein PTPRJ and then induced platelet aggregation to reduce joint function.

Platelet adhesion in type 2 diabetes: impact of plasma albumin and mean platelet volume.

BACKGROUND: Altered mean platelet volume (MPV) and plasma albumin has been reported in type 2 diabetes (T2D). MPV is suggested to predict cardiovascular risk but there is a lack of evidence for associations between MPV and platelet adhesion. Plasma albumin and magnesium are other factors reported to influence thrombotic risk. The objectives of this study were to assess the association between platelet adhesion and plasma factors with a potential role to affect platelet activation. METHODS: Blood was collected from 60 T2D patients and 60 healthy controls. Platelet adhesion to different protein surfaces induced by various soluble activators were measured in microplates. MPV, albumin and magnesium were analysed together with additional routine tests. RESULTS: Despite normal levels, plasma albumin significantly correlated with adhesion of T2D platelets but not with controls. There was a significant association between MPV and platelet adhesion in both groups, but association was smaller in T2D. Levels of glucose, HbA1c or magnesium did not correlate with platelet adhesion. CONCLUSIONS: Plasma albumin was associated with platelet adhesion in T2D suggesting that albumin may be a factor to consider upon cardiovascular risk assessment. MPV was more associated with the level of platelet adhesion in healthy individuals than in well-controlled T2D patients.

Kinetics of platelet adhesion to a fibrinogen-coated surface in whole blood under flow conditions.

AIM: To test a novel method of assessment of platelet adhesion to a fibrinogen-coated surface in whole blood under flow conditions. METHODS: We developed a fluidic device that mimics blood flow in vessels. The method of detection of platelet adhesion is based on recording of a scattered laser light signal from a fibrinogen-covered surface. Testing was performed in platelet-rich plasma (PRP) and whole blood of healthy volunteers. Control measurements were performed, followed by tests with inhibition of platelet GPIIa/IIIb and GPIb receptors. Then, the same testing sequence was performed in whole blood of persons with autoimmune thrombocytopenia and type 3 von Willebrand disease. RESULTS: The change in intensity of scattered light was 2.7 (2.4; 4.1) times higher in whole blood (0.2 ± 0.08V, n = 7) than in PRP (0.05 ± 0.02 V, n = 7), p < 0.01. The blocking of GP IIb/IIIa receptors decreased the intensity of scattered light to 8.5 (6.5;12)%; the blocking of GPIb receptors decreased it to 34 (23;58)%, p < 0.01. In the whole blood of a person with autoimmune thrombocytopenia, the inhibition of GPIb receptors decreased platelet adhesion, but no effect was observed in type 3 von Willebrand disease. Inhibition of platelet GPIIb/IIIa receptors alone or combined inhibition of GPIb and GPIIb/IIIa receptors resulted in almost total suppression of adhesion in both cases. CONCLUSION: Our system effectively registers platelet adhesion to a fibrinogen-coated surface under controlled-flow conditions and may successfully be applied to the investigation of platelet adhesion kinetics.

Immune cell phenotyping in low blood volumes for assessment of cardiovascular disease risk, development, and progression: a pilot study.

BACKGROUND: Cardiovascular disease (CVD) is the leading cause of death in the world. Given the role of immune cells in atherosclerosis development and progression, effective methods for characterizing immune cell populations are needed, particularly among populations disproportionately at risk for CVD. RESULTS: By using a variety of antibodies combined in one staining protocol, we were able to identify granulocyte, lymphocyte, and monocyte sub-populations by CD-antigen expression from 500 µl of whole blood, enabling a more extensive comparison than what is possible with a complete blood count and differential (CBC). The flow cytometry panel was established and tested in a total of 29 healthy men and women. As a proof of principle, these 29 samples were split by their race/ethnicity: African-Americans (AA) (N = 14) and Caucasians (N = 15). We found in accordance with the literature that AA had fewer granulocytes and more lymphocytes when compared to Caucasians, though the proportion of total monocytes was similar in both groups. Several new differences between AA and Caucasians were noted that had not been previously described. For example, AA had a greater proportion of platelet adhesion on non-classical monocytes when compared to Caucasians, a cell-to-cell interaction described as crucially important in CVD. We also examined our flow panel in a clinical population of AA women with known CVD risk factors (N = 20). Several of the flow cytometry parameters that cannot be measured with the CBC displayed correlations with clinical CVD risk markers. For instance, Framingham Risk Score (FRS) calculated for each participant correlated with immune cell platelet aggregates (PA) (e.g. T cell PA ß = 0.59, p = 0.03 or non-classical monocyte PA ß = 0.54, p = 0.02) after adjustment for body mass index (BMI). CONCLUSION: A flow cytometry panel identified differences in granulocytes, monocytes, and lymphocytes between AA and Caucasians which may contribute to increased CVD risk in AA. Moreover, this flow panel identifies immune cell sub-populations and platelet aggregates associated with CVD risk. This flow cytometry panel may serve as an effective method for phenotyping immune cell populations involved in the development and progression of CVD.

Modeling Clot Formation of Shear-Injured Platelets in Flow by a Dissipative Particle Dynamics Method.

The regions with high non-physiological shear stresses (NPSS) are inevitable in blood-contacting medical devices (BCMDs) used for mechanically assisted circulatory support. NPSS can cause platelet activation and receptor shedding potentially resulting in the alteration of hemostatic function. In this study, we developed a dissipative particle dynamics model to characterize clot formation (platelet-collagen and inter-platelet adhesion) of NPSS-traumatized blood at a vascular injury site. A rectangular tube of 50 × 50 × 200 µm with an 8 × 8 µm collagen-coated area was modeled as a small blood vessel and perfusion with blood. Clot formation dynamics during perfusion was simulated. NPSS-traumatized blood was modeled to have more activated platelet and fewer adhesion receptors with weakened inter-platelet binding. Computational results showed that clots grew at a faster rate while the structure of the clots was less stable and collapsed more frequently for NPSS-traumatized blood compared with normal blood. The finding that NPSS-traumatized platelets could result in quicker but more easily breakable blood clots at injury sites may explain why increased risks of thrombotic and bleeding complications occurred concurrently in patients implanted with BCMDs.

In vitro study on the hemocompatibility of plasma electrolytic oxidation coatings on titanium substrates.

Passively levitated ventricular assist devices (VADs) are vulnerable to impeller-housing contact and could benefit from surface coatings that improve wear resistance. Such coatings can be manufactured by plasma electrolytic oxidation (PEO), but their suitability for blood-contact applications needs further investigation. We therefore compared blood-surface interactions of polished titanium grade 5 (Ti Gr 5), as a general VAD reference material, uncoated ground titanium grade 4 (Ti Gr 4) and two commercially available PEO coatings on Ti Gr 4. In n = 4 static platelet adhesion tests, material samples were incubated with platelet-rich plasma (PRP) and consecutively analyzed for adhesive platelets by immunofluorescence microscopy. Additionally, PRP supernatant of incubated material samples was analyzed for changes in antithrombin III and fibrinogen concentrations by turbodimetry and enzyme-linked immunosorbent assay, respectively. We could not find any significant differences between the materials in the analyzed hemocompatibility markers (P > .05). Thus, we conclude that PEO coatings might offer a similar hemocompatibility to that of polished Ti Gr 5 and uncoated Ti Gr 4. Nevertheless, future studies should investigate blood-surface interactions of PEO coatings under realistic VAD-related flow conditions to better evaluate their potential for VAD applications.

Calcium-induced dissociation of CIB1 from ASK1 regulates agonist-induced activation of the p38 MAPK pathway in platelets.

Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that regulates activation of the c-Jun N-terminal kinase (JNK)- and p38-stress response pathways leading to apoptosis in nucleated cells. We have previously shown that ASK1 is expressed in platelets and regulates agonist-induced platelet activation and thrombosis. However, the mechanism by which platelet agonists cause activation of ASK1 is unknown. Here, we show that in platelets agonist-induced activation of p38 is exclusively dependent on ASK1. Both thrombin and collagen were able to activate ASK1/p38. Activation of ASK1/p38 was strongly dependent on thromboxane A2 (TxA2) and ADP. Agonist-induced ASK1 activation is blocked by inhibition of phospholipase C (PLC) ß/γ activity or by chelating intracellular Ca2+. Furthermore, treatment of platelets with thapsigargin or Ca2+ ionophore robustly induced ASK1/p38 activation. In addition, calcium and integrin-binding protein 1 (CIB1), a Ca2+-dependent negative regulator of ASK1, associates with ASK1 in resting platelets and is dissociated upon platelet activation by thrombin. Dissociation of CIB1 corresponds with ASK1 binding to tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6) and the autophosphorylation of ASK1 Thr838 within the catalytic domain results in full activation of ASK1. Furthermore, genetic ablation of Cib1 in mice augments agonist-induced Ask1/p38 activation. Together our results suggest that in resting platelets ASK1 is bound to CIB1 at low Ca2+ concentrations. Agonist-induced platelet activation causes an increase in intracellular Ca2+ concentration that leads to the dissociation of CIB1 from ASK1, allowing for proper dimerization through ASK1 N-terminal coiled-coil (NCC) domains.

Development of anti-matrix metalloproteinase-2 (MMP-2) nanobodies as potential therapeutic and diagnostic tools.

Matrix metalloproteinase-2 (MMP-2) is an endopeptidase involved in cardiovascular disease and cancer. To date, no highly selective MMP-2 inhibitors have been identified for potential use in humans. Aim of our work was to apply the nanobody technology to the generation of highly selective inhibitors of human MMP-2 and to assess their effects on platelet function and their applicability as conjugated nanobodies. We constructed a nanobody library after immunising an alpaca with human active MMP-2 and identified, after phage display and screening, one MMP-2 inhibitory nanobody (VHH-29), able to hinder the effects of MMP-2 on platelet activation, and one nanobody not inhibiting MMP-2 activity (VHH-136) which, chemically conjugated to a fluorescent probe, allowed the detection of human MMP-2 by flow-cytometry and immune-cytochemistry. In conclusion, we have generated and characterized two new nanotechnological molecular tools for human MMP-2 which represent promising agents for the study of MMP-2 in cardiovascular pathophysiology.

Effect of Ancylostoma ceylanicum hookworm platelet inhibitor on platelet adhesion and peripheral blood mononuclear cell proliferation.

Ancylostoma ceylanicum is a zoonotic parasitic nematode that can cause iron-deficiency anemia and malnutrition in humans. A. ceylanicum hookworm platelet inhibitor (Ace-HPI) can inhibit platelet aggregation in the host to facilitate blood sucking, but whether it possesses platelet adhesion inhibitory activity or immunomodulatory role is yet unknown. To explore the effect of Ace-HPI on platelet adhesion, we expressed the recombinant protein in two competent cells, BL21 (DE3) and Rosetta-gami2 (DE3), and incubated this protein with canine platelets in a 96-well microplate. Ace-HPI was used to stimulate peripheral blood mononuclear cells (PBMC) in vitro to investigate the effect on PBMC proliferation and cytokine expression. Results showed that Ace-HPI expressed in Rosetta-gami2 (DE3) strain was mostly soluble. The inhibitory effect of this protein on platelet adhesion was relatively weak (7-8%). This protein stimulated the proliferation of PBMC and promoted the expression of Treg and Th2 cytokines, such as IL-10 and IL-13. These results lay a foundation for exploring the role of Ace-HPI in hookworm disease pathogenesis and as a candidate molecule for hookworm vaccines.

Platelet Adhesion and Thrombus Formation in Microchannels: The Effect of Assay-Dependent Variables.

Microfluidic flow chambers (MFCs) allow the study of platelet adhesion and thrombus formation under flow, which may be influenced by several variables. We developed a new MFC, with which we tested the effects of different variables on the results of platelet deposition and thrombus formation on a collagen-coated surface. METHODS: Whole blood was perfused in the MFC over collagen Type I for 4 min at different wall shear rates (WSR) and different concentrations of collagen-coating solutions, keeping blood samples at room temperature or 37 °C before starting the experiments. In addition, we tested the effects of the antiplatelet agent acetylsalicylic acid (ASA) (antagonist of cyclooxygenase-1, 100 µM) and cangrelor (antagonist of P2Y12, 1 µM). RESULTS: Platelet deposition on collagen (I) was not affected by the storage temperature of the blood before perfusion (room temperature vs. 37 °C); (II) was dependent on a shear rate in the range between 300/s and 1700/s; and (III) was influenced by the collagen concentration used to coat the microchannels up to a value of 10 µg/mL. ASA and cangrelor did not cause statistically significant inhibition of platelet accumulation, except for ASA at low collagen concentrations. CONCLUSIONS: Platelet deposition on collagen-coated surfaces is a shear-dependent process, not influenced by the collagen concentration beyond a value of 10 µg/mL. However, the inhibitory effect of antiplatelet drugs is better observed using low concentrations of collagen.

Standardization and Validation of Fluorescence-Based Quantitative Assay to Study Human Platelet Adhesion to Extracellular-Matrix in a 384-Well Plate.

Platelets play a crucial role in the immunological response and are involved in the pathological settings of vascular diseases, and their adhesion to the extracellular matrix is important to bring leukocytes close to the endothelial cells and to form and stabilize the thrombus. Currently there are several methods to study platelet adhesion; however, the optimal parameters to perform the assay vary among studies, which hinders their comparison and reproducibility. Here, a standardization and validation of a fluorescence-based quantitative adhesion assay to study platelet-ECM interaction in a high-throughput screening format is proposed. Our study confirms that fluorescence-based quantitative assays can be effectively used to detect platelet adhesion, in which BCECF-AM presents the highest sensitivity in comparison to other dyes.

Platelet-endothelial cell interactions modulate smooth muscle cell phenotype in an in vitro model of type 2 diabetes mellitus.

Platelet (PLT)-endothelial cell (EC) interaction appears to contribute to phenotypic transition of vascular smooth muscle cells (VSMCs), which play an important role in the physiological and pathological process of vascular complications in type 2 diabetes mellitus (DM2). However, the precise mechanisms by which interactions between PLTs and ECs affect VSMC phenotype have largely remained unclear. We determined the effect of diabetic PLT-EC interaction to influence VSMC migration, proliferation, and phenotypic transformation in triple-cell coculture models using the quantitative real-time PCR, Western blot, fluorescence microscopy, wound scratch assays, CCK-8 assays, and gelatin zymography assays. Our results revealed DM2 PLT-EC interaction to be associated with a significant downregulation of VSMC-specific contractile phenotypic genes and proteins, including SM22α, smooth muscle actin, Smoothelin-B, and smooth muscle-myosin heavy chain. Inversely, VSMC-specific proliferative phenotype gene and protein levels, including cyclin D1 and 2, nonmuscle myosin heavy chain B, and PCNA were in upregulation. Furthermore, the DM2-originated PLT-EC interaction promoted the expression level of transforming growth factor-ß1, and the PI3K/Akt and matrix metalloproteinase 9 signaling pathway was activated subsequently. Finally, these reactions contributed to a synthetic phenotype of VSMCs, including the proliferation, migration, and gelatinolytic activities. These findings suggest that PLT-EC interaction modulates the phenotypic transition of VSMCs between a contractile and proliferative/synthetic phenotype under diabetic conditions, conceivably providing important implications regarding the mechanisms controlling the VSMC phenotypic transition and the development of cardiovascular complications.

The Antithrombotic Function of Sphingosine-1-Phosphate on Human Adipose-Stem-Cell-Recellularized Tissue Engineered Vascular Graft In Vitro.

Adipose stem cells (ASCs) show potential in the recellularization of tissue engineerined vascular grafts (TEVGs). However, whether sphingosine-1-phosphate (S1P) could further enhance the adhesion, proliferation, and antithrombosis of ASCs on decellularized vascular scaffolds is unknown. This study investigated the effect of S1P on the recellularization of TEVGs with ASCs. Human ASCs were derived from lipoaspirate. Scaffolds were derived from human umbilical arteries (HUAs) with treatment of 0.1% sodium dodecyl sulfate (SDS) for 48 h (decellularized HUAs; DHUAs). The adhesion, proliferation, and antithrombotic functions (kinetic clotting time and platelet adhesion) of ASCs on DHUAs with S1P or without S1P were evaluated. The histology and DNA examination revealed a preserved structure and the elimination of the nuclear component more than 95% in HUAs after decellularizaiton. Human ASCs (hASCs) showed CD29(+), CD73(+), CD90(+), CD105(+), CD31(-), CD34(-), CD44(-), HLA-DR(-), and CD146(-) while S1P-treated ASCs showed marker shifting to CD31(+). In contrast to human umbilical vein endothelial cells (HUVECs), S1P didn't significantly increase proliferation of ASCs on DHUAs. However, the kinetic clotting test revealed prolonged blood clotting in S1P-treated ASC-recellularized DHUAs. S1P also decreased platelet adhesion on ASC-recellularized DHUAs. In addition, S1P treatment increased the syndecan-1 expression of ASCs. TEVG reconstituted with S1P and ASC-recellularized DHUAs showed an antithrombotic effect in vitro. The preliminary results showed that ASCs could adhere to DHUAs and S1P could increase the antithrombotic effect on ASC-recellularized DHUAs. The antithrombotic effect is related to ASCs exhibiting an endothelial-cell-like function and preventing of syndecan-1 shedding. A future animal study is warranted to prove this novel method.