Characterization of static adhesion of human platelets in plasma to protein surfaces in microplates

Abstract

Platelet adhesion is a complex and important event for prevention of blood loss after vessel injury. This study investigated fundamental adhesive mechanisms occurring in an in vitro assay developed for the measurement of static adhesion of human platelets in plasma. The aim was to gain methodological knowledge that could be used for interpretations of results from other studies using this specific assay. Involvement of adhesive receptors was investigated by the use of various antibodies as well as therapeutic drugs (abciximab, eptifibatide and tirofiban). Inhibitors of adenosine 5'-diphosphate (ADP)-receptors (cangrelor, MRS2179) and of thromboxane A 2 (TXA 2 )-signaling (BM-531) were used to estimate the role of autocrine activation. Adhesion to collagen was found to be mainly mediated by α 2 β 1 and to some extent by α IIb β 3 .Adhesion to fibrinogen was mediated by α IIb β 3 . Also, ADP-induced adhesion to albumin was dependent on α IIb β 3 . Furthermore, experiments with cangrelor and BM-531 showed that the majority of the adhesive interactions tested were dependent on ADP or TXA 2 . We conclude that the mechanisms of adhesion measured by the static platelet adhesion assay are in accordance with the current knowledge regarding platelet activation and adhesion. Despite its simplicity, we suggest that this adhesion assay could be used as a screening device for the study of the influence of various surfaces and soluble substances on platelet adhesion.

Increased adhesive properties of platelets in sickle cell disease: roles for αIIbβ3‐mediated ligand binding, diminished cAMP signalling and increased phosphodiesterase 3A activity

“…All experiments were performed in triplicate. Co‐incubation of platelets with thrombin (50 mU/ml) was used to stimulate adhesion in some assays; as conditions that restrict platelet aggregation were utilized (absence of flow/stirring, presence of Mg 2+ )(Eriksson & Whiss, 2009), minimal platelet aggregation and disposition were observed (microscopic observations) and, thus, platelets quantified were deemed to be adhered platelets and not aggregated platelets. In assays in which pharmacological inhibitors were utilized, platelets were pre‐incubated for 15 min (37°C) with inhibitors before adding to ligand‐coated wells.…”

Effects of nicotine, its metabolites and tobacco extracts on human platelet function in vitro

“…These surfaces were chosen since platelets use different adhesive mechanisms in order to adhere to different proteins. Platelet adhesion in this assay is known to be dependent on integrin α 2 β 1 for adhesion to collagen, while adhesion to fibrinogen and albumin occurs through integrin α IIb β 3 (Eriksson & Whiss 2009). …”

Enhanced platelet adhesion in essential thrombocythemia after in vitro activation

“…In contrast, weaker stimuli represented by adhesion measured on albumin resulted in comparatively few adhesion values that were higher than the control mean + 2SD. In addition, earlier studies show that secretion of ADP is important in this assay [20,21]. Thus, we propose that multiple stimuli, which most likely are of importance in vivo [42], and a long incubation time are both needed to induce secretion and activation of exhausted ET-platelets.…”

“…Platelet adhesion was measured as previously described [18,21]. Briefly, protein-coated microplates were washed twice in 0.9% NaCl by plate inversion followed by addition of 50 μL diluted PRP and 50 μL platelet activator or solvent.…”

“…Albumin was included both as a negative control for basal adhesion and because it allows the detection of LPA-induced adhesion [20]. This experimental setup also allows investigation of both α 2 β 1 -dependent adhesion to collagen and α IIb β 3 -dependent adhesion to fibrinogen and albumin [21]. Since platelet adhesion is scarcely studied in ET, our aim was to investigate if this novel assay for measurement of static platelet adhesion could be used to detect platelet abnormalities in patients with ET.…”