We investigated the role of the two calcium-binding proteins parvalbumin (PV) and calbindin D-28k (CB) in the locomotor activity and motor coordination using null-mutant mice for PV (PV−/−), CB (CB−/−) or both proteins (PV−/−CB−/−). These proteins are expressed in distinct, mainly non-overlapping populations of neurons of the central and peripheral nervous system and PV additionally in fast-twitch muscles. In a test measuring repeated locomotor activity during 18-20 days, the analysis revealed a slightly increased activity in mice lacking either protein, while the lack of both decreased the number of beams crossed during active periods. An increase in the characteristic speed during the first 8 days could be attributed to PVdeficiency, while the elimination of CB in CB−/− and double-KO mice decreased the percentage of fast movements at all time points. In the latter, additionally a reduction of the fastest speed was observed. The alterations in locomotor activity (fast movements, fastest speed) strongly correlate with the impairment in locomotor coordination in mice deficient for CB evidenced in the runway assay and the rotarod assay. The graded locomotor phenotype (CB > PV) is qualitatively correlated with alterations in Purkinje cell firing reported previously in these mice. The presence or absence of either protein did not affect the spontaneous locomotor activity when animals were placed in a novel environment and tested only once for 30 min. In summary, the lack of these calcium-binding proteins yields characteristic, yet distinct phenotypes with respect to locomotor activity and coordination.

Introduction Response to ADP P2Y12 receptor inhibition by clopidogrel can be evaluated by various techniques. Here, we compared a functional rapid point-of-care technique (PFA-P2Y®) with the degree of biochemical inhibition assessed by the VASP/P2Y12® assay. Methods Platelet response to clopidogrel was investigated in 173 patients undergoing elective intracerebral stenting (derivation cohort n=117; validation cohort n=56). High platelet reactivity (HPR) was defined as PFA-P2Y® occlusion time <106 sec or VASP/P2Y12® platelet reactivity index (PRI) >50%. Results In the derivation cohort, ROC analysis for the ability of PFA-P2Y® to detect biochemical HPR showed high specificity (98.4%) but poor sensitivity (20.0%) and a very low AUC (0.59). The VASP/P2Y12® assay revealed two coexisting platelet populations with different levels of VASP phosphorylation: a fraction of highly phosphorylated, inhibited platelets and another of poorly phosphorylated, reactive platelets. Analysis of the PFA-P2Y® curve shape revealed different types, categorized by time of occlusion (<106 sec, 106-300 sec, >300 sec) and pattern (regular, irregular, atypical). Noteworthy, curves with late occlusion and permeable curves with an irregular or atypical pattern correlated with VASP-PRI >50% and smaller sizes of the inhibited platelet sub-population. Considering the PFA-P2Y® shape of curve for the detection of HPR improved sensitivity (72.7%) and preserved specificity (91.9%), with a rather high AUC (0.823). The validation cohort confirmed the VASP/P2Y12® assay data and the usefulness of considering the PFA-P2Y® curve shape. Conclusion In patients treated with acetyl-salicylic acid and clopidogrel for 7-10 days, the VASP/P2Y12® assay reveals two coexisting sub-populations of differentially inhibited platelets, whose relative sizes predict global PRI and distinct PFA-P2Y® curve patterns. The detailed analysis of both VASP/P2Y12® and PFA-P2Y® is necessary for an optimal detection of HPR.