Elevated platelet microparticles in transient ischemic attacks, lacunar infarcts, and multiinfarct dementias

THROMBOSIS RESEARCH 72; 295-304,1993 00493848193 $6.00 + .OOPrinted in the USA. Copyright (c) 1993 Pergamon Press Ltd. All rights reserved.

ELEVATED PLATELET MICROPARTICLES IN TRANSIENT ISCHEMIC ATTACKS, LACUNAR INFARCTS, AND MULTIINFARCT DEMENTIAS Young J. Lee, Wenche Jy, Lawrence L. Horstman, Jamal Janania, Yolanda Reyes*, Roger E. Kelley* and Yeon S. Ahnl . The William J. Harrington Center for Blood Diseases, Department of Medicine, and Department of Neurology*, University of Miami; Miami, Florida, U.S.A.

(Received 10.2.1993; accepted in revised form 10.8.1993 by Editor J.L. Moake)

Abstract Platelets release microparticles (PMP) upon activation. Elevated levels of PMP were observed in patients with immune thrombocytopenic purpura (ITP), sometimes associated with a syndrome resembling transient ischemic attack (TIA), suggesting a thrombogenic potential for PMP. To determine if this association applies to TIA and other cerebrovascular accidents (CVA) without ITP, we studied PMP profiles in 71 patients with ischemic CVA: 28 with small vessel CVA (SCVA), either lacunar infarcts or TIA; 24 with large vessel CVA (LCVA); 19 with multiinfarct dementia (MID); 12 with Alzheimer’s dementia (AD); and 31 healthy controls. The mean PMP values were: MID = 3.71 f0.51; SCVA = 3.48 kO.63; LCVA = 1.97 kO.28; AD = 1.19 f0.27; controls = 0.88 H.09, (all units x 107/mL). PMP values in all groups except AD were significantly above normal (pcO.01). However, the elevation in SCVA was more marked than in LCVA (pcO.01). Administration of the calcium channel blocker, nifedipine, to 11 TIA patients reduced PMP significantly.

Key Words: platelet activation; platelet microparticles; calcium channel blocker; dementias.

TIA; lacunar infarcts;

1 Corresponding author: The William J. Harrington Center for Blood Diseases, Department of Medicine, University of Miami School of Medicine, RMSB (R-36 A), 1600 NW 10th Ave., Miami, FL 33136, USA.

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Platelet fragmentation (or vesiculation) accompanies activation by thrombin, collagen, other agonists [1,2], or complement [3]. The platelet microparticles (PMP) released have been linked to exposure of the Factor Va receptor and expression of a catalytic surface for the assembly of the prothrombinase complex [3-61. In the bleeding disorder, Scott’s syndrome, platelets are defective both in releasing PMP and in promoting coagulation1 [4]. These findings indicate that platelet fragmentation is an essential physiological component of hemostasis accompanying platelet activation, and that PMP can be a useful marker of platelet activation [7-91. We developed a flow cytometric assay of PMP in peripheral blood and observed abnormal elevations in many patients with immune thrombocytopenic purpura (ITP) [7]. It was found that elevated PMP protected thrombocytopenic patients from bleeding, despite very low platelet count. Furthermore, a subgroup of ITP patients suffering from a syndrome resembling transient ischemic attack (TIA) had markedly elevated PMP levels [7]. These observations suggest that PMP can be thrombogenic. To determine if this relationship is limited to ITP, we initiated a pilot study to investigate PMP profiles in patients with TIA and other cerebrovascular accidents (CVA) not associated with ITP [8].

METHODS

Patient Pooulation.. Seventy-one patients with the clinical diagnosis of ischemic CVA and 12 patients with Alzheimer’s disease were studied. Thirty-one healthy volunteers (11 females and 20 males with mean age of 41) served as the control group. Table I summarizes the patient population and pertinent clinical data. Brain computed tomographic (CT) scans were performed in patients with ischemic CVA to confirm the diagnosis and determine the size of cerebral infarcts (see Table 1). Patients were selected from admissions to the Neurology Service of the University of Miami/Jackson Memorial Hospital from May to November of 1991 with the diagnosis of ischemic brain infarctions. Nineteen patients with diagnosis of multiinfarct dementia (MID) and radiologic evidence of multiple old lacunar infarcts, as well as 12 patients with the diagnosis of Alzheimer’s disease (AD), were recruited from the Dementia Unit of the Miami Veterans Administration Medical Center during the same period. lschemic brain infarctions were diagnosed clinically when a focal neurologic deficit of abrupt onset did not resolve within 24 hours, was associated with hypodense areas in a CT scan of the brain, and no sources of emboli could be found. Patients with TIA were recruited from the clinics; patients with cardiac emboli, carotid or large vessel diseases were excluded. The patient population was preponderantly male (see Table 1) since much of the study was done at the Miami Veterans Administration Hospital. The prevalence of risk factors for atherosclerosis, including hypertension and diabetes mellitus, was high and comparable in all the subgroups except Alzheimer’ disease (AD). A large percentage of patients were taking drugs known to affect platelet functions (aspirin, dipyridamole or calcium channel blockers). The proportion of patients taking antiplatelet drugs was similar in all subgroups except AD.

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TABLE 1 Summary of Patient Population and Pertinent Clinical Data.

Number of Patients Male Female Mean Age Associated Disease: Diabetes Hypertension Mean platelet count* Therapy: Aspirin Ca channel blocker Dipyridamole ??

TIA/LAC (la)

LCVA (1 b)

MID (2a)

AD (2b)

28 15 13 62.5

24 11 13 54.5

19 18 1 70.0

12 12 0 73.6

9

6 17 288f25

5 15 274&l 5

0

19 242*19 16 10 10

11 17 3

7 8 2

1 1 0

1 311 f33

+ Standard error of the the mean.

Patients were classified into four subgroups for comparisons: la. lb.

2a.

2b.

LCVA (n=24): Large vessel CVA group; included all cortical and subcortical infarcts z-l.5 cm in diameter by CT scan. SCVA (n=28): Small vessel CVA group; included all subcortical infarcts cl.5 cm and TIA patients with either normal CT scans or lacunar infarcts without dementia. MID (n=l9): Multiinfarct dementia group; defined by radiologic evidence of multiple old lacunar infarcts or periventricular diffuse white matter abnormalities consistent with extensive small vessel disease. AD (n=l2): Alzheimer’s dementia group; patients with clinical history consistent with AD, and normal CT of brain (except for minimal atrophy).

All previous medications were continued during the study, including conventional antiplatelet drugs, and no new medications were introduced (see Table 1) except for the group of 11 TIA patients selected for treatment with a calcium channel blocker (nifedipine). All of these 11 had more than 2 episodes of typical symptoms of TIA of duration less than 24 hours. Nifedipine (20-60 mg/day) was administered and recurrence of TIA symptoms and PMP levels were monitored. Blood tests and PMP assays were performed during stable phases in MID and AD, and within 10 days after acute episodes of cerebral ischemia in patients with SCVA

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and LCVA. In patients with small vessel TIA, assay of PMP was done within 10 days of episodes in all but 3 cases, and was repeated at intervals after 1-6 weeks of nifedipine therapy. The TIA patients were followed clinically for 4 to 6 months for any recurrences. Phlebw. Blood samples were obtained using 21 gauge butterfly needles from the antecubital vein or other large veins in the forearm using a light tourniquet applied to the upper extremity that was immediately released after cannulation of the vein. The first 3 mL of blood were collected for complete blood count; then, samples for assay were drawn in tubes containing the anticoagulant acid-citrate-dextrose (ACD), and assayed within 3 hours. The possibility of platelet activation ex vivo was tested by repeating the assay at intervals from 10 min to 3 hrs and longer. The results demonstrad a reproducibility of flO% for up to 3 hrs, and there was tight clustering of normal control values. PMP Au. The PMP assay was previously described [7]. Briefly, platelet-rich plasma was prepared by centrifuging whole blood at 160 xg for 10 minutes. Immediately after centrifugation, one part of the supernatant (platelet-rich plasma, PRP) was mixed with three parts of phosphate buffered saline (PBS, Dulbecco’s) containing 1.3% p-formaldehyde. After 15 minutes of fixation.at room temperature, an aliquot containing 1xl 07 platelets was incubated with 5 pL FITC- conjugated monoclonal anti-glycoprotein lb (SZ2) for 30 minutes, then diluted to 1 mL with PBS and aspirated into an EPICS V flow cytometer equipped with a 5, watt laser (Coulter Electronics, Hialeah, FL). The size of platelets and microparticles was determined by forward angle light scattering (FALS) calibrated by beads from 0.15 urn to 5 urn in size (Poly Science Inc.). PMP were identified by size (FALS) and green fluorescence: particles
RESULTS

PMP levels in small and large vessel CVA, as well as MID, were significantly higher than the control group, ~~0.01. In contrast, PMP in Alzheimer’s (AD) was not higher than normal. The means and standard error (SE) of PMP were 3.71 +0.51 for MID; 3.48 M.63 for SCVA (TWLAC); 1.97 M.28 for LCVA; 1.19 +0.27 for AD; and 0.8 M.09 for control, all in units x107 /mL. Although the mean age of the control group was younger than in the patient groups, no systematic variation of PMP values with age or gender of normal controls was observed. Mean platelet counts in each group are shown in Table 1. There was no significant difference between any of the groups. PMP levels in small vs large vessel CVA are compared in Fig. la. Patients with lacunar infarcts and those with small vessel TIA were grouped together as small vessel CVA. PMP in small vessel disease (TWLAC) was significantly higher than in the LCVA group (~~0.01). PMP levels in Alzheimer’s vs multiinfarct dementia are compared in Fig. lb. PMP levels in Alzheimer’s dementia did not differ significantly from control values. Levels of PMP in MID are significantly higher than in controls or in AD (~~0.01).

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4.0. n=28

I

299

(1a.1

(1W n=19

3.0.

T

n=24

I

LCVA

TIA/LAC

I

CONTROL

ALZHEIMER’r

MID

CONTROL

FIG 1 & PMP in large vessel CVA (LCVA) and small vessel CVA including TIA and lacunar infarcts are significantly higher (~~0.01) compared to healthy controls. PMP elevation is more marked in small h PMP in Alzheimer’s and multivessel CVA than LCVA (~~0.05). infarct dementia (MID) compared with healthy controls. PMP were significantly (~~0.01) higher in MID but not in Alzheimer’s. Column height is mean value, f standard error.

To study the effects of a calcium channel blocker, recurrent TIA were selected for nifedipine therapy patients experienced dizzy spells or hypotension, dose (10 mg/day) and then gradually increased levels were assayed after l-6 weeks of therapy.

nifedipine, on PMP, 11 patients with in a dosage of 30 to 60 mg/day. If nifedipine was administered in low to a full dose (60 mg/day). PMP

Significant decreases in PMP levels (~~0.01) were found following nifedipine therapy as shown in Fig. 2. However, mean values of PMP during therapy remained significantly higher than normal controls (p
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i

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T

BEFORE

AFTER

FIG 2 PMP levels in 11 patients with TIA before and after short term nifedipine therapy. PMP levels in these patients were significantly lower after nifedipine (~~0.01). However, mean values in this group remained significantly above normal (~~0.01).

DISCUSSION

Several groups have developed PMP assays [7,1 O-l 21 and observed increased PMP in patients undergoing cardiopulmonary bypass [lO,ll], in acute respiratory distress syndrome [lo] and in stored platelets [12]. More recently, PMP levels were measured in patients with immune thrombocytopenic purpura (ITP), a disorder in which autoantibodies interact with platelets and lead to platelet destruction [13]. It was observed that elevated levels of PMP protect ITP patients from thrombocytopenic hemorrhage. Additionally, a group of ITP patients was identified who experienced TIAlike syndromes due to occlusive episodes in small vessels of the subcortical areas, as demonstrated by MRI or CAT scans. Clinical manifestations of this subgroup ranged from recurrent episodes of dizzy spells or weakness in mild cases (often misdiagnosed as hysteria or panic attacks), to coma or seizures in severe cases, and to progressive dementias in chronic cases. It was concluded that PMP are hemostatically active in ITP and, in certain clinical settings, may be thrombogenic [7-g]. Antiplatelet antibody interacting with complement on the membrane appears to lead to platelet fragmentation in ITP [14].

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The present study was undertaken to determine if the association between elevated PMP and TIA-like syndromes reported for ITP might also be found in other TIA and dementia patients with CNS vascular occlusions. This relationship was confirmed for multiinfarct dementias, lacunar infarcts and TIA associated with small vessel occlusions [8]. The correlation was weaker for large vessel infarcts. No relationship between PMP and Alzheimer’s dementia was observed. The factors responsible for platelet fragmentation in these subgroups of ischemic cerebral disorders remain to be elucidated. Early studies by electron microscopy showed the budding of vesicles from platelet plasma membranes, following the adherence of platelets to injured vessel walls [15,16]. Interaction of platelets with altered CNS vessel walls may lead to PMP elevation in subgroups of ischemic CVA. The role of platelet hyperactivation in ischemic cerebrovascular and coronary diseases has been increasingly appreciated in recent years [17]. Platelet activation associated with ischemic cerebrovascular diseases has been demonstrated by enhanced platelet aggregation [18,19], the presence of circulating platelet aggregates 119,201, increased platelet calcium [21,22], and increased secretion of platelet granular constituents [23-251. However, none of these tests has been widely used because of inconsistent results, possible influences of various medications, or cumbersome techniques. The PMP assay introduced in [7] and further applied in this report appears promising in the evaluation of possible platelet activation in patients with ischemic CVA, including lacunar infarcts, small vessel TIA, and multiinfarct dementias. PMP levels may be less affected by antiplatelet drugs (e.g. aspirin) than other tests of platelet function. Many of our subjects (except the AD group) were on anti-platelet therapy throughout the study. Nevertheless, all groups had significantly higher PMP than the normal or AD groups, implying that conventional antiplatelet drugs have limited effect on PMP generation in these disorders. This suggestion is supported by the observation that although patients placed on nifedipine during the study showed diminished PMP values, their levels remained above normal (p
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It is not certain if the PMP are themselves active in inducing thrombi, or instead reflect a pathological state of platelet activation. PMP have been shown to adhere to the subendothelium of ever-ted rabbit aorta and to facilitate adhesion of platelets to the vessel wall in animal studies [28]. Utilization of the PMP assay in cerebrovascular disorders may aid in the detection of thrombotic risk, monitoring the effects of treatment, and identifying those CVA subclasses, associated with possible platelet hyperactivation.

Acknowledgments

Supported by a grant from the American Heart Association, Florida Affiliate; Mary Beth Weiss Research Fund in Memory of Sandy Block; the Cissy & Marvin Freedman Fund; A.J & Ethel Rothenberg Fund; the Kenneth Chasen Fund; the Kathleen & Stanley Glaser Fund; and the Coulter Corporation. We are deeply indebted to the late Dr. William J. Harrington Sr. for his encouragement on this project and his recruitment of patients in this study.

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