A Model of Platelets in the Aging Organism Reveals Increased Numbers Due to Reduced Clearance and Enhanced Activatability of Gp2b/3a and P-Selectin, Resulting in a Larger Stroke Burden
Authors: Stivala Simona , Gobbato Sara , Reiner Martin F , Lüscher Thomas F , Camici Giovanni G , Beer Juerg H. , Bonetti Nicole R 
Published: November 2019

Abstract
Background and Aims
Age isakey risk factor for cardiovascular disease (CVD). The prevalence of people aged > 60 y old is projected to rise to 25 % by the year 2050, associated with a 10 % increase in CVD.
Platelets play a major role in the progression and the final pathway of CVD. Yet, little is known on the changes in platelet function due to aging per se.
Thus, we aimed to further analyze platelets in the aging organism and their putative mechanistic role in ischemia/reperfusion (I/R) brain injury.
To dissect the potentially specific aging effects from confounding factors, such as interfering medications (eg. antiplatelet drugs) and comorbidities (eg. hypertension, diabetes), we chose an animal model of young and very old mice to tackle this apparent hen and egg problem.
Methods and Results:
C57BL/6 wildtype littermates, not spontaneously developing atherosclerosis, were studied at the ages of 12 weeks (young Cohort, yoC) and > 20 months respectively (old Cohort, oldC; n=7/7).
Platelet mass was increased in the oldC in number (yoC: 791.42 x 103/µl vs. oldC: 1719.14 x 103/µl, p = 0.013) and in size (6.35 fl vs. 7.2 fl, p=0.0037). Numerically higher reticulated platelet counts were found in the young (11.31 % vs. 4.3 %, p=0.14), as determined by thiazole orange staining and flow cytometric analysis, suggesting a decreased clearance in the old as an underlying mechanism. The red blood cell count in the yoC was significantly higher than in the old (12.34 x 106/mm3 vs. 9.97 x 106/mm3, p=0.0028), whereas white blood cell counts did not differ.
Cryosections from liver and spleen stained with a rat-anti-mouse CD41 antibody revealed larger areas marked in the yoC, thus supporting the hypothesis of reduced clearance with aging (hepatic area covered 0.83 % vs. 0.27 %, p=0.014; splenic area covered 2.04 % vs. 1.04 %, p=0.012). The glycocalicin (GC) concentration as determined by ELISA was increased in the oldC (43.26 µg/ml vs. 71.4 µg/ml, p=0.0018) but after correction for the platelet count, the GC index was similar between the groups, suggesting a steady state of platelet survival at a higher count.
For functional studies of platelet receptors and activation, washed platelets were activated with thrombin (0.1 U/ml) or collagen type I (5 µg/ml), incubated with specific anti-P-selectin and anti-activated integrin aIIbb3 (JON/A) antibodiesand analyzed by flow cytometry. While the baseline receptor expression between groups was similar, we observed an increased GP IIb/IIIa (fold increase 2.41 vs. 8.26, p=0.04) and numerically also P-selectin (fold increase 7.4 vs. 18.1, p=0.08) exposure after activation with thrombin in the oldC (Fig.1).
The baseline surface expression of GPVI and GPIb was similar between groups before and upon activation.
To study, whether the observed changes could alter stroke size and functional outcome after I/R brain injury, we studied the model of transient middle cerebral artery occlusion (tMCAO), in which a silicone-covered thread is introduced into the ICA and blocks cerebral blood flow for 30 minutes followed by 48 h of reperfusion. In a preliminary set of experiments (n=3/4), stroke size was analyzed using triphenyltetrazolium chloride (TTC) staining and revealed a doubling of the stroke volumes (21.15 mm3 vs. 43.32 mm3, p=0.20) in the old mice. This finding related to a significantly poorer neurological outcome after 48 h, as demonstrated by the RotaRod test, measuring the latency to fall from a rotating rod (55.17 s vs. 9.83 s, p = 0.0015) and by the Bederson test, a global assessment of neurological function using a scale from 0-4 (1 vs. 1.83, p=0.0036) (Fig.2) .
Summary & Conclusions:
Platelet studies in our aging model reveal 1) higher platelet numbers and larger platelet size not related to reticulated platelets, 2) a reduced hepatic and splenic clearance and 3) an increased procoagulant potential (activatability of GPIIb/IIIa), as well as a strong tendency to a pro-inflammatory response (P-selectin expression) upon thrombin exposure. These obviously acquired changes in platelet function and number may in part be responsible for 4) larger stroke sizes and poorer functional outcome observed in old mice, overall pointing towards a genuine, primarily age dependent procoagulant platelet phenotype. The model will help in delineating the specific role of platelets in age-related CVD and allow to test specific molecular or pharmacological interventions, targeting the platelet changes with aging.

Disclosures
No relevant conflicts of interest to declare.