Microfluidic whole blood testing of platelet response to pharmacological agents

Abstract

Platelets present a number of intracellular and transmembrane targets subject to pharmacological modulation, either for cardiovascular disease reduction or as an unintended drug response. Microfluidic devices allow human blood to clot on a defined surface under controlled hemodynamic and pharmacological conditions. The potencies of a number of antiplatelet and anti-cancer drugs have been tested with respect to platelet deposition on collagen under flow. Inhibitors of cyclooxygenase-1 (COX-1) reduce platelet deposition, either when added ex vivo to blood or ingested orally by patients prior to testing. Some individuals display a functional “aspirin-insensitivity” in microfluidic assay. When certain nonsteroidal anti-inflammatory drugs (NSAIDs) are taken orally, they block COX-1 acetylation by aspirin with concomitant reduction of aspirin efficacy against platelets in microfluidic assay. Both P2Y1 and P2Y12 inhibitors reduce platelet deposition under flow, as do NO donors and iloprost that target the guanylate cyclase and the prostacyclin receptor, respectively. In a microfluidic assay of 37 kinase inhibitors, dasatinib had potent antiplatelet activity, while bosutinib was less potent. Dasatinib and bosutinib have known profiles against numerous kinases, revealing overlapping and non-overlapping activities relevant to their unique actions against platelets. Also, dasatinib caused a marked and specific inhibition of GPVI signaling induced by convulxin, consistent with a dasatinib-associated bleeding risk. Microfluidic devices facilitate drug library screening, dose-response testing, and drug-drug interaction studies. Kinase inhibitors developed as anticancer agents may present anti-platelet activities that are detectable by microfluidic assay and potentially linked to bleeding risks.

Acquired platelet antagonism: off‐target antiplatelet effects of malignancy treatment with tyrosine kinase inhibitors

“…For bosutinib (which also targets Src), bleeding has also been reported , although in patients it did not affect platelet aggregation in response to several agonists . In microfluidic whole blood testing of platelet thrombus formation, bosutinib was found to be less potent than dasatinib . Overall, it appears that the BCR–ABL inhibitors used for leukemia treatment have an inhibitory effect on platelet functions, with the possible exception of nilotinib.…”

“…For bosutinib (which also targets Src), bleeding has also been reported [59], although in patients it did not affect platelet aggregation in response to several agonists [52]. In microfluidic whole blood testing of platelet thrombus formation, bosutinib was found to be less potent than dasatinib [53].…”

“…Studies with blood from patients or healthy subjects showed impairment of platelet aggregation by dasatinib given in vivo or in vitro . In particular, platelet aggregation in response to collagen, arachidonic acid and epinephrine was affected , along with collagen‐induced thrombus formation , suggesting interference with GPVI signaling and other pathways. The mechanism of dasatinib‐induced inhibition in platelets was elucidated from changes in the protein phosphorylation profile after collagen stimulation; dasatinib appeared to interfere with SFK, PLC and PI3K activities .…”

In vitro flow based systems to study platelet function and thrombus formation: Recommendations for standardization: Communication from the SSC on Biorheology of the ISTH

“…It is also ideally suited to evaluate the inhibitory potential of novel antiplatelet agents . As the assay requires only milliliter volumes of blood, this minimizes the expenditure of pharmacological agents required per assay . Moreover, the more recent development in microfluidic flow devices with even smaller channel dimensions has further reduced the required blood volume and enables screening of multiple antiplatelet agents and analysis of blood from laboratory animals.…”

Acquired alteration in platelets

“…This is of translational interest, since also with human blood, flow chamber tests are considered as a suitable initial screening tool for evaluating the efficacy of established and novel antithrombotic drugs. 53,54 Altogether, the findings of this thesis point to multiple interacting roles of the different blood components -platelets, red blood cells and plasma -in hemostatic reactions from platelet aggregation and thrombus formation to fibrin clot formation. Regarding platelets, it has become clear that different populations are formed with distinct properties and distinct interactions with other blood components.…”