Platelets, March 2012; 23(2): 116–120 Copyright ß 2012 Informa UK Ltd. ISSN: 0953-7104 print/1369-1635 online DOI: 10.3109/09537104.2011.593654
ORIGINAL ARTICLE
Low-density platelet populations demonstrate low in vivo activity in sporadic Alzheimer disease ¨ REMO1, M. MILOVANOVIC1, C. BULLER1, S. NILSSON2, & B. WINBLAD3 P. JA
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1
Department of Internal Medicine, The Vrinnevi Hospital, Norrko¨ping, Sweden, 2Department of Medical and Health Sciences, Division of Community Medicine/General Practice, Linko¨ping University, Linko¨ping, Sweden, and 3 Department of Neurobiology, KI-Alzheimer’s Disease Research Center, Karolinska Institute, Care Sciences and Society (NVS), Huddinge, Sweden
Abstract Platelets contain a substantial quantity of amyloid-precursor protein (APP) and -amyloid. However, despite the large importance of APP and -amyloid to dementia, little is known about platelets in sporadic Alzheimer dementia (AD). Furthermore, platelet heterogeneity influences human pathology and has been described to affect the progression of AD. This study investigated AD platelets with respect to density diversity and in vivo activity associated with density subfractions. We included 39 AD patients and used, as controls, 22 elderly individuals without apparent memory disorder. A continuous PercollTM gradient covering the density span 1.04–1.09 kg/l provided the basis to divide platelets of whole blood into density fractions (n ¼ 16). All platelet populations were evaluated accordingly. Platelet counts were determined electronically. A flow-cytometer was put to use to measure surface-bound fibrinogen as a measure of platelet in vivo activity. Samples obtained from patients diagnosed with sporadic AD contained platelets (fractions numbers 4–16) that circulated with significantly less surface-bound fibrinogen, i.e., their platelet activation in vivo was reduced, compared with controls. In particular, highly significant differences (p < 0.001) were obtained for the six less dense platelet populations (fractions numbers 11–16) when comparing sporadic AD with controls. In contrast, the densest AD platelets in fractions numbers 1–3 did not differ significantly from control cells with respect to in vivo platelet-bound fibrinogen. It is concluded that sporadic AD is characterized by lower density platelet populations that, while circulating, exhibited reduced activation. The clinical significance of this finding is unclear but these results suggest the importance of platelet heterogeneity in dementia as a topic for further investigation.
Keywords: Fibrinogen, dementia, platelet heterogeneity, platelet in vivo activation, sporadic Alzheimer disease
Introduction Heterogeneity of human platelets is well-established. Platelet density (kg/l), for example, is substantially diverse. Cell organelles, such as -granules and dense bodies, are determinants of platelet buoyant density [1]. Also, intracellular glycogen and mitochondria are closely dependent on density [2]. High-density platelets adhere more rapidly to collagen [3] and are associated with elevated metabolism [4]. Recent basic research from our laboratory demonstrates that high- and low-density platelets circulate in an activated state [5, 6]. Platelet heterogeneity has an impact upon human pathology. For example, small high-density platelets are a feature of active inflammatory bowel disease [7] and low-peak platelet density is a characteristic of
essential thrombocythemia [8]. Furthermore, preeclampsia severity is related to larger platelets having lower density [9]. Brains of Alzheimer disease (AD) sufferers are characterized by deposition of -amyloid in plaques. The peptide is toxic for the surrounding tissue. Recent evidence suggests that the substance damages erythrocytes [10]. Outside the central nervous system, platelets are a significant source of both amyloid-precursor protein (APP) and -amyloid [11–13]. Platelets possess all the enzymatic equipment required to process APP metabolites. Disturbances of these mechanisms in platelets are known to occur early in dementia [14, 15] and are associated with declining cognition [16]. Upon secretion of -granules, activated platelets
Correspondence: P. Ja¨remo, Department of Internal Medicine, The Vrinnevi Hospital, S-601 82 Norrko¨ping, Sweden. Tel: þ4611125662. Fax: þ4611125662. E-mail:
[email protected] (received 22 April 2011; revised 29 May 2011; accepted 31 May 2011)
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Platelet heterogeneity and Alzheimer disease release -amyloid [17, 18]. An early study found functional platelet alterations in AD [19] and a recent single report describes lower platelet activity, as estimated from soluble platelet activity markers, to be a feature of AD [20]. Platelet activity seems to be associated with cognitive decay [21]. In contrast, other scientists argue that platelet activity, as estimated from thromboxane B2 in urine is persistently augmented in AD [22]. Studies of platelet heterogeneity in the most common form of AD, the sporadic type, are rare, though coat platelets constitute an exception [23]. They are created in the laboratory after the dual provocation with collagen and thrombin. After stimulation, they express more surfacebound -granule proteins, including APP [24]. Coat platelets are associated with cognitive decline and AD dementia severity [25] and with a rapid transgression of mild cognitive impairments to manifest AD [26]. This study addressed platelet-density diversity in the sporadic form of AD. Samples of platelets in whole blood were separated by density into 16 subfractions, enabling comparison of platelet heterogeneity between AD patients with a suitable control group. This approach enables the investigation of different characteristics associated with circulating platelet populations separated by density and compared for level of activation in vivo.
Methods Study subjects This study was approved by the Institutional Review Board of Linko¨ping University Hospital. Participants gave their informed consent without exception. Demographic characteristics are described in Table I. Thirty-nine patients (age 70 8(SD) years) (20 females/19 males) were recruited. All met the clinical criteria for probable AD on the basis of diagnostic assessment by the responsible geriatrician. The AD-diagnosis was made according to clinical criteria DSM-IV. A CTscan was used to exclude brain tumors and previous major cerebral infarction. No exclusion criteria were applied. Twenty-two elderly subjects (age 66 9(SD) years) (10 females/12 males) lacking obvious memory problems served as controls. Analytical procedures Corpuscular elements of 10 ml citrate-anticoagulated whole blood were divided by density using a linear TM Percoll (General Electric Healthcare Bio-Sciences AB, Sweden) gradient covering the density span 1.04–1.09 kg/l in a 50 ml test tube [5, 6, 26]. To avoid in vitro platelet activity, the PercollTM solutions
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Table I. Demographic data for the two study groups. Sporadic AD Subjects (n) Age (years) (M, SD) Sex (f/m) Alcohol problems (n) ACE-inhibitors (n) A2-blockers (n) aˆ-blockers (n) Ca2þ-blockers (n) Diuretics (n) Statins (n) Aspirin (n) Diabetes (n) Heart disease (n) Hypertension (n) Acetylcholine esterase inhibitors (n)
Controls
p-value
39 70 8 20/19 3 3 1 9 4 5 3 4 1 8 16 17
22 66 9 10/12 0 6 5 6 7 3 6 3 0 1 14
0.040 NS NS NS NS NS NS NS NS NS NS NS NS
Note: ACE, angiotensin-converting enzyme; NS, not significant.
contained EDTA and prostaglandin E1 [6, 27]. The gradient was centrifuged for 1 hours at 2767 g. Subsequently, the bottom of the test tube was perforated and the gradient divided by gravidity into 2 ml fractions (n ¼ 16). Platelet counts were determined electronically. In all density fractions, a Beckman Coulter EPICS XL-MCLTM Flow Cytometer (Beckman Coulter, Inc., CA., USA) was used to measure platelet exterior bound fibrinogen [28]. Platelets were identified with a phycoerythrinconjugated antibody against GPIb (Dako AS, Denmark). Fibrinogen was recognized with a fluorescein isothiocyanate-conjugated chicken antihuman polyclonal antibody (Biopool AB, Sweden). The chi-square and the unpaired Student’s t-test were applied. The level of significance was set at p < 0.05.
Results Demographical data are given in Table I. The AD group proved to be older (p ¼ 0.040) and were more frequently prescribed acetyl cholinesterase inhibitors. Review of the history in the medical chart indicated that three demented participants had previous alcohol problems. The prevalence of heart disease and hypertension was unaffected by dementia, and not surprisingly then, drug prescriptions did not differ significantly between the study groups (Table I). The platelet-density distributions from both AD and control groups did not differ significantly (Figure 1), suggesting that AD did not affect platelet-density diversity. Figure 2 shows the in vivo activity of platelets in the 16 density sub-fractions. Results showed that AD was associated with significantly less surface-bound fibrinogen in most (n ¼ 13) platelet
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P. Ja¨remo et al. Platelet density distribution Platelet counts
Alzheimer
( X10 9 /L)
Controls
200
100
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0 1
2
3
4
5
6
7
8
9
10
11
12
High density platelets
13
14
15
16
Low density platelets
Figure 1. Platelet distribution across gradients is shown for both sporadic Alzheimer patients and controls. Fraction 1 contained the highest platelet density.
Platelet bound fibrinogen Alzheimer Controls
(% fibrinogen positive cells)
80
