Thrombin Receptor-activating Peptide Research Articles

Direct IL-6 inhibition prevents arterial thrombosis by reducing collagen-mediated platelets activation

Abstract Introduction Interleukin 6 (IL-6) has a pivotal role in atherothrombosis. Yet, targeting IL-6 to prevent atherothrombotic events still requires validation of efficacy and safety. The recent phase 2 RESCUE trial reported that direct inhibition of IL-6 ligand by the monoclonal antibody (mAb) Ziltivekimab is safe in patients with elevated cardiovascular risk. Purpose This project investigated the effects of direct IL-6 inhibition on arterial thrombosis, and assessed the underlying cellular mechanisms. Methods Three month old C56Bl/6 male mice received very-low dose LPS i.p. for 4 weeks. During the last week they were randomized to receive in addition either anti-mouse IL-6 mAb 200 μg i.p. every other day or IgG1 isotype control. Then thrombosis of left common carotid artery (LCCA) was induced by laser injury under anesthesia. Platelets of treated mice were isolated and stimulated with either adenosine di-phosphate (ADP), collagen-related peptide (CRP) or thrombin receptor activating peptide (TRAP). Platelets activation was measured by flow-cytometry, as expression of P-selectin and JON/A.The effect of direct IL-6 inhibition was confirmed in patients with stable coronary artery disease (sCAD) by ex-vivo incubation of isolated platelets with either anti-human IL-6 mAb 1.5 μg/mL or IgG1 isotype control. Platelet activation was measured by flow-cytometry, as expression of P-selectin. Results Mice treated with anti-IL6 antibody displayed a significantly longer time-to-occlusion after the LCCA (Figure 1), without any significant difference in coagulation parameters. After stimulation with CRP, platelets were significantly activated in control mice, but not in mice treated with anti-IL6 mAb. Similarly, activation of isolated platelets from patients with sCAD was significantly reduced (Figure 2) by the ex-vivo treatment with anti-IL6 mAb. Conclusion The direct inhibition of IL-6 reduces platelets reactivity to collagen, thereby blunting arterial thrombosis upon endothelial damage. These results provide a potential mechanism to explain the reduction of cardiovascular risk associated to direct IL-6 inhibition. Conclusion The direct inhibition of IL-6 reduces platelets reactivity to collagen, thereby blunting arterial thrombosis upon endothelial damage. These results provide a potential mechanism to explain the reduction of cardiovascular risk associated to direct IL-6 inhibition.Figure 1Figure 2

Single-cell mass cytometry analysis unveils prothrombotic expression and heterogeneity in essential thrombocythemia

Abstract Background/Introduction Essential thrombocythemia (ET) is recognized for its hallmark elevated platelet count and a consequent increased risk of thromboembolic events including coronary events. Despite its clinical significance, the pathophysiological mechanisms predisposing ET patients to thrombosis remain largely undefined. Purpose The aim of this pioneering study was to shed light on the increased thrombotic risk in ET by conducting a detailed phenotypical characterization of platelet expression using single-cell mass cytometry, comparing ET patients to age- and sex-matched healthy controls. Methods Platelet samples from six patients diagnosed with ET and six healthy individuals were analyzed. This analysis utilized an extensive panel of 18 transmembrane regulators pivotal for platelet function and activation. Measurements were taken both at baseline and after ex-vivo stimulation with thrombin receptor-activating peptide (TRAP). Advanced clustering analysis via the FlowSOM algorithm helped identify specific subclusters of platelets with a prothrombotic expression profile. Results Our investigation revealed a significant overexpression of the activation marker CD62P (P-Selectin, p=0.049) and of the collagen receptor GPVI (p=0.044) in non-stimulated ET platelets (Figure 1A-C). Conversely, a notable decrease in expression was observed for the fibrinogen receptor GPIIb/IIIa units CD41 (p=0.036) and CD61 (p=0.044), as well as for the von Willebrand factor receptor CD42b (p=0.044). The FlowSOM analysis distinguished two prothrombotic subclusters of ET platelets, particularly highlighting one cluster, which was significantly overrepresented in ET (22.13% of total ET platelets versus 2.94% in controls, p=0.035, Figure 1D-E). Conclusion This study presents compelling evidence of a distinct prothrombotic phenotype in ET, marked by significant alterations in platelet expression profiles. The overexpression of CD62P and GPVI, alongside the underexpression of CD41, CD61, and CD42b, underscores the complex pathophysiological landscape of ET. Importantly, we identified a specific subcluster, particularly overrepresented in ET, with a specific prothrombic signature. Our findings, while only hypothesis generating, propose GPVI as a potential therapeutic target to mitigate thrombosis in ET patients, paving the way for targeted interventions in this high-risk population.

Platelet Function, Platelet Size and Content of Reticulated Platelets: Interactions in Patients Receiving Dual Antiplatelet Therapy.

Increased platelet activity is a risk factor of thrombotic events in cardiovascular patients. We studied the relationship between platelet function, platelet size, and the content of reticulated platelets (RP) in patients with coronary heart disease (CHD, n = 55) and acute coronary syndrome (ACS, n = 95) receiving acetylsalicylic acid + clopidogrel or ticagrelor, respectively. The control group consisted of patients with risk factors for CHD, but with no CHD/ACS and free of antiplatelet drugs (n = 66). Platelet function was evaluated by the exposure of activated glycoprotein (GP) IIb-IIIa and P-selectin. In the control group, platelets were activated by TRAP (Thrombin Receptor Activating Peptide) 10 µM, and ADP 20, 5, 2.5 µM, and in the CHD/ACS groups, by TRAP 10 µM, and ADP 20 5 µM (±epinephrine 20 µM). Platelet size was assessed by the mean volume, % large forms, and forward scattering. RP were stained by thiazole orange. In the control group, activated GP IIb-IIIa and P-selectin correlated with platelet size and RP content after platelet activation by all agonists. Despite the decrease in platelet activity by antiplatelet drugs, most correlations (primarily for activated GP IIb-IIIa) were preserved in the CHD/ACS patients. In conclusion, increased platelet size and RP content are associated with increased platelet activity and the reduced efficacy of antiplatelet therapy.

The differential formation and composition of leukocyte-platelet aggregates induced by various cellular stimulants

BackgroundLeukocyte-platelet aggregates comprise a pathogenic link between hemostasis and immunity, but the prerequisites and mechanisms of their formation remain not understood. AimsTo quantify the formation, composition, and morphology of leukocyte-platelet aggregates in vitro under the influence of various cellular activators. MethodsPhorbol-12-myristate-13-acetate (PMA), lipopolysaccharide (LPS), thrombin receptor-activating peptide (TRAP-6), and adenosine diphosphate (ADP) were used as cellular activators. Flow cytometry was utilized to identify and quantify aggregates in whole human blood and platelet-rich plasma. Cell types and cellular aggregates were identified using fluorescently labeled antibodies against the appropriate cellular markers, and cell activation was assessed by the expression of appropriate surface markers. For confocal fluorescent microscopy, cell membranes and nuclei were labeled. Neutrophil-platelet aggregates were studied using scanning electron microscopy. ResultsIn the presence of PMA, ADP or TRAP-6, about 17–38 % of neutrophils and 61–77 % of monocytes formed aggregates with platelets in whole blood, whereas LPS did not induce platelet aggregation with either neutrophils or monocytes due the inability to activate platelets. Similar results were obtained when isolated neutrophils were added to platelet-rich plasma. All the cell types involved in the heterotypic aggregation expressed molecular markers of activation. Fluorescent and electron microscopy of the aggregates showed that the predominant platelet/leukocyte ratios were 1:1 and 2:1. ConclusionsFormation of leukocyte-platelet aggregates depends on the nature of the cellular activator and the spectrum of its cell-activating ability. An indispensable condition for formation of leukocyte-platelet aggregates is activation of all cell types including platelets, which is the restrictive step.

Phenolic profile, cheminformatics, and antiplatelet aggregation activity of orange and purple sweet potato (Ipomoea batatas L.) storage roots

Sweet potatoes are rich in cardioprotective phytochemicals with potential anti-platelet aggregation activity, although this benefit may vary among cultivars/genotypes. The phenolic profile [HPLC-ESI(−)-qTOF-MS2], cheminformatics (ADMET properties, affinity toward platelet proteins) and anti-PA activity of phenolic-rich hydroalcoholic extracts obtained from orange (OSP) and purple (PSP) sweet potato storage roots, was evaluated. The phenolic richness [Hydroxycinnamic acids> flavonoids> benzoic acids] was PSP > OSP. Their main chlorogenic acids could interact with platelet proteins (integrins/adhesins, kinases/metalloenzymes) but their bioavailability could be poor. Just OSP exhibited a dose-dependent anti-platelet aggregation activity [inductor (IC50, mg.ml−1): thrombin receptor activator peptide-6 (0.55) > Adenosine-5′-diphosphate (1.02) > collagen (1.56)] and reduced P-selectin expression (0.75–1.0 mg.ml−1) but not glycoprotein IIb/IIIa secretion. The explored anti-PA activity of OSP/PSP seems to be inversely related to their phenolic richness. The poor first-pass bioavailability of its chlorogenic acids (documented in silico) may represent a further obstacle for their anti-PA in vivo.

Binding of coagulation factor IXa to procoagulant platelets revisited: Low affinity and interactions with other factors

Binding of activated factor IX (fIXa) to the phosphatidylserine-expressing procoagulant platelets is a critical step in blood coagulation, which is necessary for the membrane-dependent intrinsic tenase complex assembly and factor X activation. However, the nature and parameters of the fIXa binding sites on the procoagulant platelet surface remain unclear. We used flow cytometry to elucidate the quantitative details of the fluorescently labeled fIXa binding to gel-filtered activated platelets. FIXa bound to the procoagulant platelet subpopulation only, with the parameters (maximal number of binding sites at 58900 ± 3400, Kd at 1000 ± 170 nM) similar to binding observed with phospholipid vesicles. No specific high-affinity binding sites for fIXa were detected, and binding proceeded similarly for different methods of procoagulant platelet production (thrombin, thrombin receptor activation peptide, collagen-related peptide, their combinations, or calcium ionophore A23187). Factor VIII, known to form a high affinity complex with fIXa, enhanced fIXa binding to platelets. In contrast, only competition effects were observed for factor X, which binds fIXa with much lower affinity. Unexpectedly, fIXa itself, fIX, and prothrombin also dose-dependently enhance fIXa binding at concentrations below 1000 nM, suggesting the formation of membrane-bound fIXa dimers and fIXa-prothrombin complexes on platelets. These findings provide a novel perspective on the fIXa binding site on procoagulant platelets, which does not have any major differences from pure phospholipid-based model membranes, exhibits inherently low affinity (3–5 orders of magnitude below the physiologically relevant fIXa concentration) but is significantly enhanced by its cofactor VIII, and regulated by previously unknown membrane interactions.

Improving platelet function following prophylactic platelet transfusion in patients with hematological malignancies.

Platelet transfusion is a standard treatment to prevent bleeding in patients with hematological malignancies. Although transfusions can improve platelet count, their impact on platelet function remains controversial. We conducted flow cytometry to assess platelet function before and after transfusion and performed subgroup analyses to examine differences based on blood type, corrected count increment (CCI), and platelet microparticles. Overall, 50 patients who received prophylactic platelet transfusion were enrolled. CD42b expression increased, whereas CD41 expression decreased after transfusion. Apheresis platelets exhibited the lowest expression of PAC-1 and P-selectin when exposed to agonist stimulations. PAC-1 expression increased under high adenosine diphosphate (ADP) stimulation, while P-selectin expression increased under both high ADP and thrombin receptor-activating peptide stimulation. In the subgroup analysis, patients with a CCI >4500 and those with the same blood types exhibited a more significant increase in PAC-1 and P-selectin expression under agonist stimulation. When comparing apheresis platelets collected on different days, only the percentage of platelet-derived microparticles showed a significantincrease. Prophylactic transfusion improved platelet function. Platelet function significantly improved in patients with a CCI >4500, those with the same blood types as that of apheresis platelets, or those with platelet-derived microparticle levels <4.7%. No significant improvement in platelet function was noted after the transfusion of different blood types with acceptable compatibility or the transfusion of incompatible blood types. Our results suggest that transfusing platelets with the same blood type remains the optimal choice.

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

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. 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. 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. 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.

Platelet Activation Pathways Controlling Reversible Integrin αIIbβ3 Activation.

Background Agonist-induced platelet activation, with the integrin αIIbβ3 conformational change, is required for fibrinogen binding. This is considered reversible under specific conditions, allowing a second phase of platelet aggregation. The signaling pathways that differentiate between a permanent or transient activation state of platelets are poorly elucidated. Objective To explore platelet signaling mechanisms induced by the collagen receptor glycoprotein VI (GPVI) or by protease-activated receptors (PAR) for thrombin that regulate time-dependent αIIbβ3 activation. Methods Platelets were activated with collagen-related peptide (CRP, stimulating GPVI), thrombin receptor-activating peptides, or thrombin (stimulating PAR1 and/or 4). Integrin αIIbβ3 activation and P-selectin expression was assessed by two-color flow cytometry. Signaling pathway inhibitors were applied before or after agonist addition. Reversibility of platelet spreading was studied by microscopy. Results Platelet pretreatment with pharmacological inhibitors decreased GPVI- and PAR-induced integrin αIIbβ3 activation and P-selectin expression in the target order of protein kinase C (PKC) > glycogen synthase kinase 3 > β-arrestin > phosphatidylinositol-3-kinase. Posttreatment revealed secondary αIIbβ3 inactivation (not P-selectin expression), in the same order, but this reversibility was confined to CRP and PAR1 agonist. Combined inhibition of conventional and novel PKC isoforms was most effective for integrin closure. Pre- and posttreatment with ticagrelor, blocking the P2Y 12 adenosine diphosphate (ADP) receptor, enhanced αIIbβ3 inactivation. Spreading assays showed that PKC or P2Y 12 inhibition provoked a partial conversion from filopodia to a more discoid platelet shape. Conclusion PKC and autocrine ADP signaling contribute to persistent integrin αIIbβ3 activation in the order of PAR1/GPVI > PAR4 stimulation and hence to stabilized platelet aggregation. These findings are relevant for optimization of effective antiplatelet treatment.

Ex Vivo Antiplatelet Effects of Oral Anticoagulants.

The impact of non-vitamin K antagonist oral anticoagulants (NOACs) on platelet function is still unclear. We conducted a comprehensive ex vivo study aimed at assessing the effect of the four currently marketed NOACs on platelet function. We incubated blood samples from healthy donors with concentrations of NOACs (50, 150 and 250 ng/mL), in the range of those achieved in the plasma of patients during therapy. We evaluated generation of thrombin; light transmittance platelet aggregation (LTA) in response to adenosine diphosphate (ADP), thrombin receptor-activating peptide (TRAP), human γ-thrombin (THR) and tissue factor (TF); generation of thromboxane (TX)B2; and expression of protease-activated receptor (PAR)-1 and P-selectin on the platelet surface. All NOACs concentration-dependently reduced thrombin generation compared with control. THR-induced LTA was suppressed by the addition of dabigatran at any concentration, while TF-induced LTA was reduced by factor-Xa inhibitors. ADP- and TRAP-induced LTA was not modified by NOACs. TXB2 generation was reduced by all NOACs, particularly at the highest concentrations. We found a concentration-dependent increase in PAR-1 expression after incubation with dabigatran, mainly at the highest concentrations, but not with FXa inhibitors; P-selectin expression was not changed by any drugs. Treatment with the NOACs is associated with measurable ex vivo changes in platelet function, arguing for antiplatelet effects beyond the well-known anticoagulant activities of these drugs. There are differences, however, among the NOACs, especially between dabigatran and the FXa inhibitors.

Platelet Microparticle-Derived MiR-320b Inhibits Hypertension with Atherosclerosis Development by Targeting ETFA.

Hypertension and atherosclerosis often occur simultaneously. This study aimed to explore the role and mechanism of platelet microparticle (PMP) -derived microRNA-320b (miR-320b) in patients with hypertension accompanied by atherosclerosis.We collected samples from 13 controls without hypertension and atherosclerosis and 20 patients who had hypertension accompanied by atherosclerosis. In vitro, platelets were activated by Thrombin receptor-activating peptide to produce PMPs. HUVECs were induced by CoCl2 to mimic a hypoxic environment in vitro. RT-qPCR was employed to detect the expression levels of CD61, miR-320b, and ETFA. The protein expression level of ETFA was evaluated via Western blotting. Furthermore, 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, 5-ethynyl-2'-deoxyuridine, and wound healing assays were employed to assess the proliferation and migration of HUVECs. Enzyme-linked immunosorbent assay was used to measure the oxidative stress and inflammation-related factor expression.The expression of miR-320b was reduced in both platelets and PMPs but increased in plasma. MiR-320b promoted CoCl2-induced HUVEC viability, proliferation, and migration. The levels of the oxidative stress factors SOD and GSH as well as the inflammatory factor IL-10 were elevated in the CoCl2 + miR-320b mimics group compared with both the CoCl2 + mimics NC and CoCl2 groups. Conversely, the levels of the oxidative stress factors MDA and ROS as well as the inflammatory factors IL-6, TNF-α, and IL-1β were decreased. These results were regulated by miR-320b targeting ETFA.PMP-derived miR-320b inhibits the development of hypertension accompanied by atherosclerosis by targeting ETFA.

Acute and subacute effects of strenuous exercise on platelet aggregation, coagulation and fibrinolysis in patients with stable coronary artery disease

IntroductionStrenuous exercise may occasionally cause coronary thrombosis with myocardial infarction and sudden cardiac death. Materials and methodsPatients with stable coronary artery disease (CAD) (n = 164) and healthy individuals (n = 25) performed strenuous exercise on a bicycle ergometer. Blood was drawn at baseline, immediately after exercise and 2 h later. Platelet aggregation was measured with Multiplate® Analyzer. Thrombin generation was determined using a thrombogram and by measuring prothrombin fragment 1 + 2 (F1 + 2). A clot lysis assay was used to investigate fibrinolysis. ResultsFrom baseline to immediately after exercise, thrombin receptor activating peptide (TRAP)-induced platelet aggregation increased in CAD patients (Δ77 AU × min, 95 % confidence interval (CI): 46;107) and in healthy individuals (Δ153 AU × min, 95%CI: 75;232). Endogenous thrombin potential (ETP) was unaffected by exercise, whilst F1 + 2 increased (Δ17%, 95%CI: 11;24) in CAD patients. Fibrin clot lysis time increased by 9 % (95%CI: 1–17) in CAD patients and by 26 % (95%CI: 8;45) in healthy individuals. When comparing baseline to 2 h post-exercise, TRAP-induced platelet aggregation remained slightly elevated in both CAD patients (Δ53 AU × min, 95%CI: 22;84) and healthy individuals (Δ140 AU × min, 95%CI: 62;219). In contrast, ETP and F1 + 2 decreased in CAD patients (Δ-6 %, 95%CI: −10;-1 and Δ-8 %, 95%CI: -14;-2). Moreover, clot lysis time decreased (Δ-19 %, 95%CI: −27;-11) in patients with CAD and returned to baseline in healthy individuals. All p-values were <0.05. ConclusionsPlatelet aggregation and F1 + 2 were substantially elevated immediately after exercise in CAD patients, indicating a pro-thrombotic state. After 2 h of recovery, they exhibited a markedly increase in fibrinolysis. Similar results were observed in healthy individuals.

Association of Impedance Aggregometry-Measured Platelet Aggregation With Thromboembolic Events in Patients Who Undergo Carotid Endarterectomy: A Pilot Study

ObjectivesThe aim of the current study was to assess the relationship between thrombin receptor activator peptide 6 (TRAP test), adenosine-5′-diphosphate (ADP test) and arachidonic acid (ASPI test) and stroke/TIA, using the multiple electrode aggregometry (Multiplate®) in patients undergoing CEA. DesignA retrospective study. SettingVascular surgery operating rooms of university hospital. ParticipantsOne hundred and thirty-one out of 474 patients undergoing CEA between November 2020 and October 2022. InterventionsNone. Measurements & Main ResultsA pre-operative blood sample of all enrolled patients was analyzed using the multiplate analyzer. Receiver operating characteristics curves were generated to test the ability of TRAP, ADP and ASPI in discriminating perioperative thromboembolic stroke/TIA. A logistic LASSO regression model was used to identify factors independently associated with stroke/TIA. Eight patients suffered of perioperative stroke/TIA. Although all the platelet functional assays showed an excellent predictive performance an ADP value exciding 72 U showed the highest specificity (87%) and sensitivity (68%) in discriminating patients who had perioperative thromboembolic stroke/TIA with a negative predictive value of 99% and a positive predictive value of 15%. After LASSO regression an ADP> 72 U and need of shunt during CEA were the only two variables independently associated with perioperative stroke/TIA. ConclusionSince the ADP test was independently associated with perioperative stroke/TIA, the assessment of platelet reactivity using Multiplate® may offer potential utility in monitoring patients undergoing CEA.

Soluble urokinase plasminogen activator receptor, platelet aggregation, and carotid plaque thickness in diabetes: A cross-sectional analysis

BackgroundSoluble urokinase plasminogen activator receptor (suPAR) is an inflammatory biomarker strongly linked with cardiovascular disease in diabetes. By investigating its association with platelet aggregation levels and carotid plaque thickness, we can potentially improve the characterization of cardiovascular pathophysiology in type 1 (T1D) and type 2 diabetes (T2D). MethodsSuPAR was measured post-hoc in plasma collected in two cross-sectional studies including 1) individuals with T1D and 2) individuals with T2D without known cardiovascular disease. Platelet aggregation was analyzed employing impedance aggregometry using the agonists arachidonic acid (ASPI), adenosine diphosphate (ADP), and thrombin receptor activating peptide-6 (TRAP). Carotid plaque thickness was assessed bilaterally, and the thickest plaque (cPTmax) was analyzed. Correlations and associations of suPAR with platelet aggregation and cPTmax were assessed with Pearson's R and linear regressions adjusted for sex, age, C-reactive protein, estimated glomerular filtration rate, and platelet levels (only for aggregation analysis). ASPI analyses were only performed in participant not receiving aspirin treatment. Standardized estimates with 95 % confidence intervals are presented. ResultsTwo-hundred eighty-three with T1D (mean age 55 years, 44 % women) and 493 individuals with T2D (mean age 65 years, 33 % women) were included. SuPAR levels were positively correlated to platelet aggregation in both T1D (ADP: R = 0.18, p = 0.003; ASPI R = 0.13, p = 0.026) and T2D (ADP R = 0.14, p = 0.002; ASPI R = 0.14, p = 0.017). In adjusted linear regressions higher SuPAR levels were associated with higher ADP induced platelet aggregation in both T1D (26 (8, 44) AU/min, p = 0.004) and T2D (22 (3, 42) AU/min, p = 0.025), while associations to the other agonists were not conclusive. Higher suPAR levels were associated with thicker cPTmax in T1D (0.19 (0.07, 0.32) mm, p = 0.003), but not in T2D (p = 0.94). ConclusionHigher levels of suPAR, an inflammatory generalized disease biomarker, were associated with increased platelet aggregation and thicker cPTmax, in type 1 diabetes. Similar associations with platelet aggregation were found in type 2 diabetes, but not for carotid plaque thickness. These results suggest distinct cardiovascular pathomechanisms in T1D and T2D, although prospective studies are needed to confirm these findings.

Anti-PF4/Heparin Monoclonal Antibodies Induce Thrombus Formation in an Ex Vivo Endothelialized Microfluidic System

Introduction: Heparin-induced thrombocytopenia (HIT) is a serious adverse reaction to heparin. Antibodies against platelet factor 4 (PF4) or a heparin/PF4 complex have been reported to activate platelets, neutrophils and possibly endothelial cells, resulting in thrombocytopenia, hypercoagulability and thromboembolic complications. While the impact of anti-PF4/heparin antibodies on platelet, neutrophil and monocyte activation has been extensively studied, the role of endothelial cells in HIT-associated thrombosis remains underexplored. In particular, the interactions between HIT-induced procoagulancy and endothelial cells under dynamic conditions is not completely analyzed. In this report, we investigated how HIT antibodies induce thrombosis in an endothelialized microfluidic system. Methods: Fibrinogen microfluidic channels were coated with confluent monolayer of human umbilical vein endothelial cells (HUVECs). Cells were primed with low-dose TNF-α to induce a sub-thrombotic endothelial activation, prior to perfusion of whole blood. Recalcified unstimulated or Thrombin Receptor Activator Peptide 6 (TRAP-6) activated whole blood was perfused at a venous flow rate. HIT-thrombosis model was established and tested with monoclonal anti-PF4/heparin antibodies in whole blood. In brief, whole blood was pre-incubated with unfractionated heparin (UFH, 0.2 IU/mL and 100 IU/mL). Next, anti-PF4/heparin antibodies were introduced to the whole blood mixture and incubated for 30 minutes. Whole blood was recalcified and perfused over resting or primed endothelial cells at a venous shear stress. Thrombus formation was recorded over time. Results: The endothelialized microfluidic model successfully captures sub-thrombotic conditions under venous shear. Activated platelets induced a procoagulant shift and three-dimensional thrombi. We applied our thrombosis model to investigate thrombus formation induced by a HIT mimicking monoclonal antibody. We show that the heparin-dependent platelet activation predicts the thrombotic response in the microfluidic system: HIT antibodies in absence of heparin did not exert a pro-thrombotic effect on activated endothelial cells. Co-incubation with 0.2 IU/mL UFH induced thrombosis, embolization of thrombus material and channel occlusion only when endothelial cells were primed with TNF-α. The pro-thrombotic effect of HIT antibodies was fully reversed at the super-therapeutic dose of 100 IU/mL UFH. Conclusions: HIT antibodies induce thrombosis and embolization in a system emulating physiological environment. For the first time, we present a comprehensive thrombosis model that incorporates the established thrombogenicity of HIT-mimicking antibodies. We show that our thrombosis model incorporates both endothelial- and blood-based control of thrombosis. Primed endothelial cells and antibody-induced procoagulancy trigger thrombosis in a concerted fashion, allowing investigation of the underlying mechanisms and possible preclinical evaluation of potential inhibitors of HIT-thrombosis. Figure 1, upper panel: Maximal thrombus surface area coverage of TNF-α primed endothelial cells, perfused with unstimulated, TRAP-6 activated or HIT-like IgG/heparin challenged whole blood. HIT-like IgG did not increase thrombus formation in absence of heparin. Addition of low-dose heparin exerted a strong pro-thrombotic effect, that was fully reversible with a super-therapeutic concentration of heparin. Lower panel: representative images of thrombi formed by HIT-like IgG in absence and under low or high-dose heparin. Platelets: green, Calcein-AM. Scale bar: 200 µm.

Platelets from Ehlers-Danlos Syndrome Patients Exhibit Reduced GPVI Levels and Impaired Integrin αIIbβ3 inside-out Signaling

Background: Patients with Ehlers-Danlos syndrome (EDS) have a high susceptibility to bleeding. Easy bruising is included as a criterion for the classification of multiple types of EDS. Although the association between EDS and the tendency to bleed has been acknowledged for a long time, a definitive understanding of the underlying mechanisms contributing to bleeding diathesis in EDS patients is still lacking. Objective: To characterize platelet function in a cohort of EDS patients and a murine EDS model. Methods: A cohort of 53 patients with hypermobile (49%), classical (26%), classical-like (21%), or vascular (4%) types of EDS and a group of 53 healthy control subjects matched by age and gender were included in the study in accordance with the University of Iowa Institutional Review Board-approved protocol. The International Society of Thrombosis and Haemostasis bleeding assessment tool (ISTH-BAT) was used to assess the bleeding tendency in study participants. Because a single mutation in hypermobile type is currently unknown, we used Col5a 1 +/− mice as a murine model of the classical type of EDS. The function of human and mouse platelets was evaluated using standardized agonist-induced in vitro assays. Results: The mean ISTH-BAT score was 0.1 in healthy controls and 9.2 in EDS patients ( P &amp;lt; 0.001). An abnormal ISTH-BAT score was observed in 33 out of 53 (62%) EDS patients and 0 of 53 healthy controls ( P &amp;lt; 0.001). Platelets from EDS patients exhibited reduced aggregation and αIIbβ3 activation (with normal αIIbβ3 surface exposure) in response to stimulation with collagen, collagen-related peptide, or thrombin receptor activator peptide-6, compared to healthy controls. Platelet secretion of dense and α-granules and surface phosphatidylserine exposure were comparable between EDS and healthy subjects. Evaluation of collagen receptors on the surface of resting platelets showed a mild deficiency of GPVI in EDS patients compared to healthy controls ( P &amp;lt; 0.001), while the integrin α2 levels were similar in both groups ( P = 0.152). Analysis of GPVI signaling revealed downregulation of Syk (Y525/526), PLCγ2 (Y1217), and talin (S425) phosphorylation in EDS patients ( P &amp;lt; 0.05 vs. healthy controls), suggesting a defect in collagen-induced integrin αIIbβ3 inside-out signaling. Consistent with our observations in EDS patients, Col5a1 +/− mice demonstrated a bleeding tendency characterized by prolonged tail-bleeding time ( P &amp;lt; 0.05 vs. wild-type control). Platelet function testing showed that Col5a 1 +/− mouse platelets recapitulated the abnormalities detected in human EDS platelets. Conclusions: Our data corroborate the observation that EDS patients exhibit a high risk of hemorrhagic complications.EDS platelets are characterized by reduced baseline GPVI expression that was associated with decreased agonist-induced platelet aggregation, αIIbβ3 activation, and impaired collagen-induced integrin αIIbβ3 inside-out signaling.

In vitro comparison of platelet aggregation between zalunfiban and selatogrel in healthy donors

Abstract Background Two subcutaneously injected antiplatelet drugs, zalunfiban and selatogrel, are under development for prehospital administration for STEMI and (N)STEMI, respectively. Zalunfiban is a glycoprotein IIb/IIIa inhibitor that produces high-grade inhibition of platelet aggregation induced by ADP or a thrombin receptor activating peptide (TRAP) within 15 minutes. Selatogrel is a P2Y12 inhibitor that produces high-grade inhibition of platelet aggregation initiated by ADP within 15 minutes. We have studied the relative potencies of the drugs at levels achievable in vivo in inhibiting platelet aggregation. Since trisodium citrate (TSC) has been reported to enhance the antiplatelet effect of zalunfiban and inhibit the antiplatelet effect off selatogrel, aggregation studies were performed with both blood anticoagulated with TSC and the non-chelating anticoagulant PPACK. Whereas both ADP and thrombin are present at the site of vascular injury within seconds, we tested the effects of both ADP and TRAP. Purpose To compare directly the effects of zalunfiban and selatogrel on ADP- and TRAP-induced platelet aggregation. Methods Light transmission platelet aggregometry was performed within 4 hours of blood collection (BioData PAP-8E). Whole blood from healthy donors not taking aspirin (N=10) was anticoagulated with 3.2% TSC and 100 μM PPACK. Platelet-rich plasma (PRP) was prepared and adjusted to a platelet count of 250,000/µl. Three clinically relevant incremental concentrations of zalunfiban and selatogrel were added to PRP and platelet aggregation was initiated with 20 μM ADP or 20 μM TRAP. Tracings were recorded for at least 10 minutes after agonist addition. Platelet aggregation was quantified based on the instrument’s measurement of the primary slope (PrSl) of aggregation, which is a measure of the change in light transmission per unit time sustained over at least a 15 second period. Results In TSC-anticoagulated PRP, zalunfiban was more effective in inhibiting the PrSl of both ADP- and TRAP-induced platelet aggregation at all drug concentrations [Figure 1, panel A and B]. In PPACK-anticoagulated PRP, selatogrel showed greater inhibition of ADP-induced platelet aggregation at the lowest concentration tested, similar inhibition at the intermediate concentration, and slightly less inhibition at the highest doses. In sharp contrast, with TRAP-induced aggregation, zalunfiban demonstrated dose-related inhibition, reaching greater than 80% inhibition at the highest dose, whereas selatogrel produced less than 20% inhibition at all three doses. Conclusion Selatogrel is a potent inhibitor of ADP-induced platelet aggregation, but has low impact on TRAP-induced platelet aggregation, whereas zalunfiban is a potent inhibitor of both ADP and TRAP-induced platelet aggregation. The difference between the drugs needs to be considered in light of the known generation of thrombin at the site of vascular injury within seconds.Comparison of Platelet Inhibition

Apoptotic Pathway Inhibition in Cold-Stored Platelet Concentrates Facilitates Retained Ability to Form Thrombi

Introduction: Platelet concentrates (PCs) are used to treat or prevent bleeding in patients with impaired platelet function or after injury. However, the standard storage of PCs at room-temperature (RT) increases the risk of bacterial contamination and is associated with reduced platelet functionality due to storage lesions. Our group reported that cold-stored PCs have better functionality compared to RT-stored PCs but reduced in vivo circulation time due to cold-induced apoptosis. In order to investigate cold-stored PCs hemostatic functionality with and without apoptosis inhibition, we established an ex vivo model simulating physiological conditions of blood flow to evaluate the contribution of cold-stored PCs in thrombus formation. Methods: Apheresis-derived PCs were stored for 1, 4, 7 and 10 days under agitation at 4°C or RT either with or without the apoptosis inhibitor G04 (RhoA GTPase inhibitor). Next, PCs were stained with CD41 antibody, recalcified and incubated with TRAP6 (thrombin receptor-activating peptide 6) to initiate thrombus formation. Platelet-depleted whole blood samples from healthy donors were spiked-in with PCs and administered to the microfluidic channels of the ex vivo system (Bioflux), under physiological shear pressure. After five minutes of perfusion the resulting thrombi were imaged and analyzed. Ability to form thrombi was further assessed by thromboelastography and impedance aggregometry. Results: Platelets from 24h storage PCs showed stable thrombus formation upon activation with TRAP6. Unstimulated platelets did not form any thrombi during the entire period of perfusion. To evaluate the effect of cold storage on platelet functions we analyzed the extent of thrombus formation of PCs stored at RT and 4°C upon CD41 staining. Our results indicate that cold-stored platelets form larger clots under flow conditions and maintain higher functionality compared to PCs stored at RT. Moreover, incubation of cold-stored PCs with the apoptosis inhibitor G04 improved ex vivo thrombus formation compared to PCs stored without the inhibitor, without inducing any adverse effects (Figure 1). Conclusion: The results indicate that our ex vivo assay, which simulates PC transfusion in thrombocytopenic patients, is suitable to test the hemostatic functions of PCs under physiological flow conditions. We demonstrated that PCs stored at 4°C show better thrombus formation ability upon stimulation by agonists compared to RT-stored PCs and that the ability to form clots under shear stress is still preserved after incubation with the apoptosis inhibitor. To conclude, cold-storage of PCs in combination with apoptosis inhibition is a promising stragedy to reduce the risk of bacterial contamination and prolong in vivo survival time while preserving platelet's hemostatic functionality.

Increased Responsiveness of Stored Platelets after Short-Term Refrigeration

Introduction: Refrigeration of platelets is considered to provide advantages in therapy of acute hemorrhage due to increased platelet responsiveness. The alleviation of inhibitory signaling caused by cold temperature (CT) has been identified as an important mechanism contributing to enhanced platelet reactivity, detectable in freshly prepared platelets within 1 h of cold storage. The aim of this study was to confirm the effects of short-term refrigeration in platelets from apheresis-derived platelet concentrates (APC). Methods: APC were stored under standardized conditions for 1 day or for 2 days at room temperature and then refrigerated for 1 h, followed by sampling of platelets for analysis. Platelet reactivity was measured by aggregation studies using threshold concentrations of different agonists and by detection of fibrinogen binding using flow cytometry. The exploration of inhibitory signaling comprised the detection of VASP phosphorylation using flow cytometry or Western blot and the measurement of cyclic nucleotide levels. Results: Aggregation responses induced with ADP, collagen, or thrombin receptor-activating peptide-6 (TRAP-6) were increased in APC after cold storage for 1 h, associated with elevated TRAP-6-induced fibrinogen binding. VASP phosphorylation levels were decreased after cold exposition, detectable in 1-day- and 2-day-stored APC with flow cytometry, and in 2-day-stored APC with Western blot technique. Induced cGMP levels were lower after storage at CT in APC on day 1 and on day 2, whereas cAMP levels were reduced on 2-day-stored APC. Conclusion: Short-term refrigeration for 1 h is sufficient to induce an attenuation of inhibitory signaling, accompanied with increased aggregation responses in APC stored for up to 2 days. The “on demand” refrigeration of PC may be a reasonable approach for the preparation of platelets with enhanced responsiveness to treat patients with hemorrhage more effectively, which should be further addressed in consecutive studies.

Decreased Platelet Aggregation in Patients with Decompensated Liver Cirrhosis and TIPS Implantation.

Transjugular intrahepatic portosystemic shunt (TIPS) implantation is an effective treatment of portal hypertension in patients with decompensated liver cirrhosis. However, some patients develop TIPS thrombosis with recurrence of portal hypertension. The role of platelets in TIPS thrombosis and the necessity of antiplatelet therapy is unclear. Therefore, we aimed to study platelet function in patients with liver cirrhosis prior to and after TIPS implantation. Platelet aggregation was tested in peripheral and portal-vein blood patient samples on the day (D) of TIPS implantation (D0), D4 and D30 following the procedure (platelet count above 100 × 103/µL, aspirin starting on D5) using whole-blood impedance aggregometry (WBIA) and light transmission aggregometry (LTA). In addition, surface platelet activation markers (P-selectin, activated GPIIb/IIIa) and platelet-neutrophil complexes (PNCs) were assessed by flow cytometry. Thrombin receptor activating peptide 6 (TRAP-6), adenosine diphosphate (ADP) and arachidonic acid (AA) were used as agonists. Healthy subjects were included as controls. Agonist-induced platelet aggregation was reduced (WBIA: TRAP-6 p < 0.01, ADP p < 0.01, AA p < 0.001; LTA: TRAP-6 p = 0.13, ADP p = 0.05, AA p < 0.01) in patients (D0, n = 13) compared with healthy subjects (n = 9). While surface activation markers at baseline were negligibly low, the percentage of PNCs was higher in patients than in controls (p < 0.05). ADP-induced P-selectin expression was increased (p < 0.001), whereas TRAP-6-induced GPIIb/IIIa activation was impaired (p < 0.001) in patients versus controls. PNC formation in response to agonists was not different between groups. Results did not differ between peripheral and portal-vein blood of patients (D0, n = 11) and did not change over time (D0, D4, D30) following TIPS implantation (n = 9). In summary, patients with decompensated liver cirrhosis display in vitro platelet aggregation defects in response to various agonists. Defective aggregation persists upon TIPS implantation. Therefore, we conclude that antiplatelet treatment to prevent TIPS thrombosis is questionable.