Role of Tyrosine Kinase Syk in Thrombus Stabilisation at High Shear

Understanding the pathways involved in the formation and stability of the core and shell regions of a platelet-rich arterial thrombus may result in new ways to treat arterial thrombosis. The distinguishing feature between these two regions is the absence of fibrin in the shell which indicates that in vitro flow-based assays over thrombogenic surfaces, in the absence of coagulation, can be used to resemble this region. In this study, we have investigated the contribution of Syk tyrosine kinase in the stability of platelet aggregates (or thrombi) formed on collagen or atherosclerotic plaque homogenate at arterial shear (1000 s−1). We show that post-perfusion of the Syk inhibitor PRT-060318 over preformed thrombi on both surfaces enhances thrombus breakdown and platelet detachment. The resulting loss of thrombus stability led to a reduction in thrombus contractile score which could be detected as early as 3 min after perfusion of the Syk inhibitor. A similar loss of thrombus stability was observed with ticagrelor and indomethacin, inhibitors of platelet adenosine diphosphate (ADP) receptor and thromboxane A2 (TxA2), respectively, and in the presence of the Src inhibitor, dasatinib. In contrast, the Btk inhibitor, ibrutinib, causes only a minor decrease in thrombus contractile score. Weak thrombus breakdown is also seen with the blocking GPVI nanobody, Nb21, which indicates, at best, a minor contribution of collagen to the stability of the platelet aggregate. These results show that Syk regulates thrombus stability in the absence of fibrin in human platelets under flow and provide evidence that this involves pathways additional to activation of GPVI by collagen.

How Much Does the Platelet Aggregate Influence the Total Leukocyte Count? Comparison Between Manual and Automated Impedance Methods in Domestic Cats

Background: Automated hematology analyzers have been developed to optimize the time between analyses and have promising precision and accuracy. Complete blood count (CBC) is often requested as part of veterinary clinical examination. Automated analyzers are often used to determine CBCs, since processing as well as container-related errors may occur owing to variable sizes, aggregates, white or red blood cell fragments, and effects of EDTA on cell morphology. Platelet aggregates frequently occur in felines, with studies reporting a prevalence of approximately 71%. The aim of the present study was to evaluate the influence of exercise aggregates on the global white blood cell count of domestic cats using automated hematological counters with the impedance method.Materials, Methods & Results: Blood samples of 140 cats, irrespective of age, sex, and breed, were collected into EDTA-containing tubes. The samples were obtained via routine clinical examinations at the Veterinary Hospital of the Federal Rural University of Rio de Janeiro (UFRRJ) and processed at the Veterinary Parasitology Experimental Chemotherapy Laboratory (LQEPV), belonging to the same institution. All the samples were processed on the Sysmex pocH-100iV Diff automated hematology apparatus according to the manufacturer's recommendations. Leukocyte counts were also manually determined using a duplicate Neubauer chamber. Standard dilutions were prepared immediately after the automated analysis. To identify the occurrence of platelet aggregates, a blood smear was made and visualized under a brightfield microscope at a magnification of 10× and scored 0 to 3 (G1, G2, G3, and G4) based on the aggregation intensity. In case of changes, the groups were subdivided according to the intensity of occurrence. Of the 140 samples analyzed, 76.4% (107/140) showed some degree of platelet aggregation. The maximum variation in leukocyte counts determined by the automatic and the manual technique in G1 was 2,500 cells. In G2, it was possible to identify a variation of 6,500 nucleated cells, whereas in G3, this value was 7,100 cells. In G4, where platelet aggregation was intense, the variation between counts was up to 15,000 nucleated cells. A significant difference of variation in total white blood cell count between manual and automated methods was observed when compared to animals that did not show any degree of platelet aggregation (P < 0.05). Of the total samples, 23.57% (33/140) comprised G1, 24.28% (34/140) G2, 22.14% (31/140) G3, and 30% (42/140) G4. Of the 140 samples analyzed, 107 showed aggregates, pseudo-thrombocytopenia, and changes in the total number of leukocytes.Discussion: Samples with higher platelet aggregate formation showed greater interference in global leukometry when analyzed using the hematological counter. White blood cell counts determined by automated analyzers should be interpreted with caution and compared to manual counts when there is significant platelet aggregation in the sample. The findings reinforce the importance of reconfirming the results obtained using an automated equipment in order to avoid misinterpretations that may influence diagnosis and therapy. It is essential to re-check the values obtained from an automated equipment with traditional methods in order to minimize possible errors generated by the equipment, since such errors may affect the clinical diagnosis and subsequently, the therapeutic approach chosen.

Study of Reversible Platelet Aggregation Model by Nonlinear Dynamics

Blood cell platelets form aggregates upon vessel wall injury. Under certain conditions, a disintegration of the platelet aggregates, called “reversible aggregation”, is observed in vitro. Previously, we have proposed an extremely simple (two equations, five parameters) ordinary differential equation-based mathematical model of the reversible platelet aggregation. That model was based on mass-action law, and the parameters represented probabilities of platelet aggregate formations. Here, we aimed to perform a nonlinear dynamics analysis of this mathematical model to derive the biomedical meaning of the model’s parameters. The model’s parameters were estimated automatically from experimental data in COPASI software. Further analysis was performed in Python 2.7. Contrary to our expectations, for a broad range of parameter values, the model had only one steady state of the stable type node, thus eliminating the initial assumption that the reversibility of the aggregation curve could be explained by the system’s being near a stable focus. Therefore, we conclude that during platelet aggregation, the system is outside of the influence area of the steady state. Further analysis of the model’s parameters demonstrated that the rate constants for the reaction of aggregate formation from existing aggregates determine the reversibility of the aggregation curve. The other parameters of the model influenced either the initial aggregation rate or the quasi-steady state aggregation values.

Circulating MicroRNAs and Monocyte–Platelet Aggregate Formation in Acute Coronary Syndrome

Background Monocyte–platelet aggregates (MPAs) are a sensitive marker of in vivo platelet activation in acute coronary syndrome (ACS) and associated with clinical outcomes. MicroRNAs (miRs) play an important role in the regulation of platelet activation, and may influence MPA formation. Both, miRs and MPA, could be influenced by the type of P2Y12 inhibitor. Aim To study the association of platelet-related miRs with MPA formation in ACS patients on dual antiplatelet therapy (DAPT), and to compare miRs and MPA levels between prasugrel- and ticagrelor-treated patients. Methods and Results We analyzed 10 circulating platelet-related miRs in 160 consecutive ACS patients on DAPT with low-dose aspirin and either prasugrel (n = 80) or ticagrelor (n = 80). MPA formation was measured by flow cytometry without addition of platelet agonists and after simulation with the toll-like receptor (TLR)-1/2 agonist Pam3CSK4, adenosine diphosphate (ADP), or arachidonic acid (AA). In multivariate regression analyses, we identified miR-21 (β = 9.50, 95% confidence interval [CI]: 1.60–17.40, p = 0.019) and miR-126 (β = 7.50, 95% CI: 0.55–14.44, p = 0.035) as independent predictors of increased MPA formation in vivo and after TLR-1/2 stimulation. In contrast, none of the investigated miRs was independently associated with MPA formation after stimulation with ADP or AA. Platelet-related miR expression and MPA formation did not differ significantly between prasugrel- and ticagrelor-treated patients. Conclusion Platelet-related miR-21 and miR-126 are associated with MPA formation in ACS patients on DAPT. miRs and MPA levels were similar in prasugrel- and ticagrelor-treated patients.

Gain-of-function variant p.Pro2555Arg of von Willebrand factor increases aggregate size through altering stem dynamics

The multimeric plasma glycoprotein von Willebrand factor (VWF) is best known for recruiting platelets to sites of injury during primary hemostasis. Generally, mutations in the VWF gene lead to loss of hemostatic activity and thus the bleeding disorder von Willebrand Disease. By employing cone and platelet aggregometry and microfluidic assays, we uncovered a platelet glycoprotein (GP)IIb/IIIa-dependent prothrombotic gain-of-function (GOF) for variant p.Pro2555Arg, located in the C4-domain, leading to an increase in platelet aggregate size. We performed complementary biophysical and structural investigations using circular dichroism spectra, small angle X-ray scattering, NMR spectroscopy, molecular dynamics simulations on the single C4-domain and dimeric wildtype and p.Pro2555Arg constructs. C4-p.Pro2555Arg retained the overall structural conformation with minor populations of alternative conformations exhibiting increased hinge flexibility and slow conformational exchange. The dimeric protein becomes disordered and more flexible. Our data suggest that the GOF is not affecting the binding affinity of the C4-domain for GPIIb/IIIa. Instead, the increased VWF dimer flexibility enhances temporal accessibility of platelet binding sites. Using an interdisciplinary approach, we revealed that p.Pro2555Arg is the first VWF variant, which increases platelet aggregate size and show a shear-dependent function of the VWF stem region, which can become hyperactive through mutations. Prothrombotic GOF variants of VWF are a novel concept of a VWF-associated pathomechanism of thromboembolic events, which is of general interest to vascular health but which is not yet considered in diagnostics. Thus, awareness should be raised for the risk they pose. Furthermore, our data implicate the C4-domain as a novel anti-thrombotic drug target.

Biorheology of occlusive thrombi formation under high shear: in vitro growth and shrinkage

Occlusive thrombi formed under high flow shear rates develop very rapidly in arteries and may lead to myocardial infarction or stroke. Rapid platelet accumulation (RPA) and occlusion of platelet-rich thrombi and clot shrinkage have been studied after flow arrest. However, the influence of margination and shear rate on occlusive clot formation is not fully understood yet. In this study, the influence of flow on the growth and shrinkage of a clot is investigated. Whole blood (WB) and platelet-rich plasma (PRP) were perfused at high shear rates (> 3,000 s−1) through two microfluidic systems with a stenotic section under constant pressure. The stenotic section of the two devices are different in stenotic length (1,000 vs 150 μm) and contraction angle of the stenosis (15° vs 80°). In all experiments, the flow chamber occluded in the stenotic section. Besides a significantly increased lag time and decreased RPA rate for PRP compared to WB (p < 0.01), the device with a shorter stenotic section and steeper contraction angle showed a shear-dependent occlusion and lag time for both PRP and WB. This shear-dependent behavior of the platelet aggregate formation might be caused by the stenotic geometry.

Platelet-to-lymphocyte and Neutrophil-to-lymphocyte Ratios Predict Target Vessel Restenosis after Infrainguinal Angioplasty with Stent Implantation

Platelet-to-lymphocyte (PLR), neutrophil-to-lymphocyte (NLR) and lymphocyte-to-monocyte (LMR) ratios are associated with the occurrence of critical limb ischemia in peripheral artery disease (PAD). We therefore investigated whether PLR, NLR or LMR are linked to target vessel restenosis (TVR) following infrainguinal angioplasty and stenting. Moreover, we studied on-treatment platelet reactivity and neutrophil-platelet aggregate (NPA) formation as potential underlying mechanisms. Platelet, neutrophil, lymphocyte and monocyte counts were determined one day after angioplasty and stenting in 95 stable PAD patients. Platelet reactivity and NPA formation in response to protease-activated receptor−1 stimulation were measured by light transmission aggregometry (LTA) and flow cytometry, respectively. PLR and NLR were significantly higher in patients who subsequently developed TVR (both p < 0.05). In contrast, LMR did not differ significantly between patients without and with TVR (p = 0.28). A PLR ≥ 91 and NLR ≥2.75 were identified as the best thresholds to predict TVR, providing sensitivities of 87.5% and 81.3%, and specificities of 34.9% and 50.8%, respectively, and were therefore defined as high PLR and high NLR. TVR occurred significantly more often in patients with high PLR and high NLR than in those with lower ratios (both p < 0.05). Patients with high PLR and high NLR exhibited significantly increased on-treatment platelet aggregation compared to those with lower ratios, and patients with high PLR had higher levels of NPA formation (all p < 0.01). In conclusion, PLR and NLR predict TVR after infrainguinal angioplasty with stent implantation. Platelet activation and neutrophil-platelet interaction may be involved in the underlying pathomechanisms

Glycoprotein VI is not a Functional Platelet Receptor for Fibrin Formed in Plasma or Blood

Glycoprotein VI (GPVI), a platelet collagen receptor, is crucial in mediating atherothrombosis. Besides collagen, injured plaques expose tissue factor (TF) that triggers fibrin formation. Previous studies reported that GPVI also is a platelet receptor for fibrinogen and fibrin. We studied the effect of anti-GPVI antibodies and inhibitors of GPVI signaling kinases (Syk and Btk) on platelet adhesion and aggregate formation onto immobilized fibrinogen and different types of fibrin under arterial flow conditions. Fibrin was prepared from isolated fibrinogen (“pure fibrin”), recombinant fibrinogen (“recombinant fibrin”), or generated more physiologically from endogenous fibrinogen in plasma (“plasma fibrin”) or by exposing TF-coated surfaces to flowing blood (“blood fibrin”). Inhibition of GPVI and Syk did not inhibit platelet adhesion and aggregate formation onto fibrinogen. In contrast anti-GPVI antibodies, inhibitors of Syk and Btk and the anti-GPIb antibody 6B4 inhibited platelet aggregate formation onto pure and recombinant fibrin. However, inhibition of GPVI and GPVI signaling did not significantly reduce platelet coverage of plasma fibrin and blood fibrin. Plasma fibrin contained many proteins incorporated during clot formation. Advanced optical imaging revealed plasma fibrin as a spongiform cushion with thicker, knotty, and long fibers and little activation of adhering platelets. Albumin intercalated in plasma fibrin fibers left only little space for platelet attachment. Pure fibrin was different showing a dense mesh of thin fibers with strongly activated platelets. We conclude that fibrin formed in plasma and blood contains plasma proteins shielding GPVI-activating epitopes. Our findings do not support a role of GPVI for platelet activation by physiologic fibrin.