Transient adhesion refractoriness of circulating platelets under shear stress: the role of partial activation and microaggregate formation by suboptimal ADP concentration

Abstract

Summary. Exposure of whole blood (WB) to subendothelial extracellular matrix (ECM) under shear stress in the cone and plate(let) analyser (CPA) results in platelet adhesion, followed by release reaction and aggregation of circulating platelets on the adherent platelets. The properties of circulating non-adhered platelets in the CPA was studied by exposure of WB to ECM at a high shear rate (1300/s) for 2 min (1st run), followed by transfer of the suspension to a new ECM-coated well for a second run (2nd run) under similar conditions. The results of the 2nd run demonstrated transient adhesion refractoriness associated with platelet microaggregate formation in the suspension. The adhesion refractoriness was dependent on platelet activation during the 1st run and was prevented by addition of apyrase (ADP scavenger) or ADP receptor inhibitor, suggesting a role for ADP in mediating this response. Furthermore, exposure of WB samples to suboptimal concentrations of ADP (0´4± 1 mmol/l) or a thrombin receptor activating peptide (TRAP) (5 mmol/l) for 2 min resulted in a similar transient platelet adhesion refractoriness to ECM under flow conditions. The transient platelet refractoriness and microaggregate formation induced by ADP was associated with a transient reduction in glycoprotein (GP)Ib, increased P-selectin expression and increased fibrinogen binding by circulating platelets. These data suggest a role for platelet agonists at suboptimal concentrations in modulating platelet function and limiting the expansion of the thrombus.

Testing agonist-induced platelet aggregation by the Impact-R [Cone and plate(let) analyzer (CPA)]

“…In the present study, the technology was extended to assess platelet aggregation in response to suboptimal concentrations of various agonists. The agonists' suboptimal concentrations used in this study were found in a pilot study to significantly decrease the SC from 11.2 AE 2.6% to the range of 1-3% due to micro-aggregate formation and platelet adhesion refractoriness [16]. Thus, the degree of decline in platelet adhesion following blood treatment with an agonist indicates the degree of platelet response to the tested agonist.…”

“…In a previous study, we demonstrated that blood exposed to ADP at sub-optimal concentration prior to the CPA test results in platelet activation and micro-aggregate formation followed by adhesion refractoriness that transiently recovered within 20-40 min [16]. This phenomenon has already been used to adapt the CPA technology in testing the platelet response to aspirin and clopidogrel treatment [17][18][19].…”

Impact – The Cone and Plate(let) Analyzer: Testing Platelet Function and Anti-Platelet Drug Response

“…The properties of circulating non-adhered platelets exposed to the released compounds (from adherent and activated platelets) were studied [6]. The results demonstrated that platelets in the suspension at the end of the Impact test form microaggregates presumably in response to the released reagents, accompanied with a reduced single platelet count.…”

A new point-of-care method for monitoring anti-platelet therapy: Application of the cone and plate(let) analyzer

“…The experimental design used to test platelet adhesion refractoriness was performed as previously described [13] with some modifications. Briefly, platelet agonists were added to 200 ml of whole blood in final concentrations of 1.25 mM (ADP) or 320 mM (arachidonic acid, AA) (DiaMed, Cressier, Switzerland).…”

“…We first described the phenomenon of transient adhesion refractoriness of circulating platelets, which can be detected by the CPA test in 2001 [13]. Sub-optimal platelet activation induced by either shear stress or an agonist results in platelet micro-aggregates formation.…”