Mechanisms Of Platelet Inhibition Research Articles

498 Bruton’s tyrosine kinase (BTK) inhibitors impede platelet aggregation but not adhesion to collagen.

OBJECTIVES/GOALS: The research objectives of this project are to elucidate the effects of Bruton’s tyrosine kinase inhibitors (BTKi) of varying target specificity on platelet function with regard to platelet aggregation, adhesion, spreading, and intracellular signaling as measured by kinase phosphorylation. METHODS/STUDY POPULATION: Blood from healthy volunteers was obtained and processed to obtain both washed platelets and platelet-rich plasma. The samples were then treated with one of the BTKi drugs or with vehicle (DMSO) at concentrations matching patient blood concentrations derived from clinical trials and pharmacokinetic studies. The incubated samples were then analyzed in an aggregometer using one of several agonists. Aggregation was stopped after five minutes with a perchloric acid-based lysis buffer. The samples were then analyzed by SDS-PAGE and immunoblotting to quantify BTK protein and BTK phosphorylation. Adhesion was assessed by incubating washed platelets treated with BTKi on microtiter wells coated with fibrinogen or collagen and quantifying adherent platelets by their endogenous acid phosphatase activity. RESULTS/ANTICIPATED RESULTS: We found that ibrutinib, zanubrutinib, and pirtobrutinib all completely inhibited collagen-induced platelet aggregation, whereas they did not inhibit aggregation induced by thrombin, ristocetin, arachidonic acid, or the PAR1 activator peptide SFLLRN (T6). Acalabrutinib inhibited collagen-induced platelet aggregation only at high concentrations (1-2 micromolar). At the lower concentration of 200 nanomolar, comparable to the concentration required for the other BTK inhibitors to completely inhibit platelet aggregation, acalabrutinib failed to inhibit aggregation but did inhibit auto-phosphorylation, indicating an impact on signaling. None of the BTKi drugs inhibited adhesion of platelets to collagen-coated surfaces. DISCUSSION/SIGNIFICANCE: Our data show the inhibitory effect of BTKi on collagen-induced platelet aggregation and signaling. However, it remains unclear whether the inhibition is due to an effect on BTK itself or other related kinases. Better insight into the mechanisms of platelet inhibition by BTKi may help guide the development of BTKi with a lower risk of hemorrhage.

Sanguinarine Attenuates Collagen-Induced Platelet Activation and Thrombus Formation.

Sanguinarine, a benzophenanthridine alkaloid, has been described to have an antiplatelet activity. However, its antithrombotic effect and the mechanism of platelet inhibition have not thoroughly been explored. The current study found that sanguinarine had an inhibitory effect on thrombus formation. This inhibitory effect was quite evident both in the flow-chamber assays as well as in a murine model of FeCl3-induced carotid artery thrombosis. Further investigations also revealed that sanguinarine inhibited the collagen-induced human platelet aggregation and granule release. At the same time, it also prevented platelet spreading and adhesion to immobilized fibrinogen. The molecular mechanisms of its antiplatelet activity were found to be as follows: 1. Reduced phosphorylation of the downstream signaling pathways in collagen specific receptor GPVI (Syk-PLCγ2 and PI3K-Akt-GSK3β); 2. Inhibition of collagen-induced increase in the intracellular Ca2+ concentration ([Ca2+]i); 3. Inhibition of integrin αIIbβ3 outside-in signaling via reducing β3 and Src (Tyr-416) phosphorylation. It can be concluded that sanguinarine inhibits collagen-induced platelet activation and reduces thrombus formation. This effect is mediated via inhibiting the phosphorylation of multiple components in the GPVI signaling pathway. Current data also indicate that sanguinarine can be of some clinical value to treat cardiovascular diseases involving an excess of platelet activation.

The Future of Antiplatelet Therapy in Cardiovascular Disease

Mechanisms of platelet inhibition are reviewed with emphasis on the pharmacokinetic and pharmacodynamic determinants of clinical efficacy and safety of antiplatelet drugs. Current developments in antiplatelet therapy are discussed in relation to both primary and secondary prevention of atherothrombotic complications. Interindividual variability in response to antiplatelet agents and new drug targets are outlined within the context of optimizing the balance between the cardiovascular benefits and bleeding risks of antiplatelet therapy. Recent advances in the pharmacogenetics of thienopyridines open the realistic prospect of a personalized choice of the most appropriate antiplatelet agent and tailored dose adjustment for an individual patient.

Anesthetic induction with ketamine inhibits platelet activation before, during, and after cardiopulmonary bypass in baboons.

The objective of this study was to investigate the effects of antifactor D monoclonal antibody (Mab) 166-32 on platelet activation during and after hypothermic cardiopulmonary bypass (CPB) in baboons. Fourteen baboons (mean weight, 15 kg) underwent hypothermic CPB. Seven of them were treated with a single injection of antifactor D Mab 166-32 (5 mg/kg) and the other seven animals were given saline as control. Each baboon was sedated with an intramuscular injection of 10 mg/kg of ketamine hydrochloride. A 20-gauge angiocatheter was placed in the cephalic vein, and 5 mg of diazepam was administered intravenously. Anesthesia was maintained with 0.80% to 2.25% isoflurane, 100% O2, and an inspiratory tidal volume of 13 mL/kg at a rate of 13 breaths per minute throughout the surgical procedure except during CPB. Pancuronium bromide, 0.1 mg/kg, was administered to achieve adequate muscle paralysis. Blood samples were collected before CPB, during CPB, and 1, 2, 3, and 6 h after CPB. Assays were performed to measure platelet activation [CD62P (P-selectin)] using immunofluorocytometric methods. There were no significant differences on CD62P expression of platelets between control and antibody groups before CPB (105 +/- 12% vs. 99 +/- 8%, P=NS), during normothermic CPB (62 +/- 6% vs. 63 +/- 19%, P=NS), during hypothermic CPB (55 +/- 8% vs. 54 +/- 13%, P=NS), and 1, 3, or 6 h after CPB (74 +/- 20% vs. 81 +/- 11%, P=NS). Anesthetic induction with ketamine caused significant reduction in the platelet activation in both groups. Ketamine did not affect complement, neutrophil, and monocyte activation or cytokine production. Further studies on the mechanisms of platelet inhibition by ketamine are warranted.

Cell-free and erythrocytic S-nitrosohemoglobin inhibits human platelet aggregation.

Nitric oxide (NO) and related molecules are thought to inhibit human platelet aggregation by raising levels of cGMP. Both oxidative stress (reactive oxygen species) and hemoglobin (Hb) seem to oppose NO effects. A major fraction of NO in the blood is bound to thiols of Hb, forming S-nitrosohemoglobin (SNO-Hb), which releases the NO group on deoxygenation in the microcirculation. Here we show that (1) both cell-free and intraerythrocytic SNO-Hb (SNO-RBC) inhibit platelet aggregation, (2) the oxidation state of the hemes in Hb influences the response--SNO-metHb (which is functionally similar to SNO-deoxyHb) has greater platelet inhibitory effects than SNO-oxyHb, and (3) the mechanism of platelet inhibition by SNO-Hb is cGMP independent. We suggest that the RBC has evolved a means to counteract platelet activation in small vessels and the proaggregatory effects of oxidative stress by forming SNO-Hb.

Inhibition of platelet function by contrast media: iopamidol and ioxaglate versus iothalamate. Work in progress.

The effects of an ionic contrast agent, meglumine iothalamate (Conray-60), and two newer low-osmolality radiographic contrast media, sodium meglumine ioxaglate (Hexabrix) and iopamidol (B-15,000), on platelet aggregation and secretion responses were studied. All three agents inhibited platelet responses during stimulation with adenosine diphosphate (ADP), epinephrine, and collagen. Platelet function was inhibited by iothalamate at concentrations of 11 mg iodine/ml and above, and by the newer agents at concentrations above 30 mg iodine/ml. Addition of exogenous calcium decreased the iothalamate-induced inhibition of aggregation but did not improve dense granule secretion. There was no consistent effect of exogenous calcium on platelet inhibition by iopamidol and ioxaglate. These studies indicate that the newer agents inhibit platelet function less than iothalamate does, and that chelation of Ca2+ may not be the major mechanism of platelet inhibition by contrast agents.