THROMBOSIS @Pergamon
RESEARCH 15;95-108 Press Ltd.1979.
Printed
in Great
Britain
oo49-3848/7g/o6ol-oog5
$02.00/o
INCREASED IN VIVO AND IN VITRO PLATELET FUNCTION IN TYPE II- AND TYPE IV-HYPERLIPOPROTEINEIA
J. Heinrich Joist, R. Kendall Baker, and Gustav Schonfeld Departments of Medicine, Preventive Medicine and Pathology, Washington University School of Medicine, St. Louis, Missouri
(Received 29.9.1978; Accepted by
in revised form 12.2.1979. Editor M.A. Packham)
ABSTRACT Platelet function in vivo and in vitro was examined in 10 patients with type IIa-, 8 patients with type IIb-, and 16 patients with type IV-hyperlipidemia (HIP) and 24 control subjects closely matched for age and sex. Patients with type IIb- and IV-HLP showed significantly shorter template bleeding times in the presence of similar blood platelet concentrations. Increased platelet factor 3-availability in intact platelet-rich plasma (PRP) and PRP exposed to collagen was observed in all patient groups. Plasma antiheparin activity was increased in patients with type IIa- a d type IV-HLP. Platelet adhesiveness, platelet aggregation and 1E C-serotonin release in response to ADP, epinephrine and collagen, as well as plasma von Willebrand factor activity, were generally not increased-in either patient group. Increased platelet function in endogenous HLP may be related to both increased platelet turnover secondary to premature atherosclerotic disease and abnormalities of platelet lipid composition induced by HLP-plasma.
INTRODUCTION There is substantial evidence that platelets may play an important role in the development and progression of premature atherosclerotic disease and its complications, such as myocardial infarction and stroke (l-3). A strong association between premature atherosclerotic disease and increased blood lipid levels has also been well recognized (4-6). This evidence and previously
Reprint Requests to: Dr. J.H. Joist, Div. of Hematology-Oncology, St. Louis University School of Medicine, 1325 S. Grand Blvd., St. Louis, MO 63104 Presented in part at the 49th Meeting of the American Heart Association, Miami Beach, Florida, November, 1976.
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reported findings of increased in vitro platelet sensitivityto aggregating and release-inducingstimuli in patients with type II-hyperlipidemia(HLP) (7) raise the question of a possible pathogeneticlink between HLP, increased platelet function and premature atheroscleroticdisease. The present study was undertaken to study the bleeding time as a measure of platelet function in vivo (8) in patients with type II- and IV-HLP and to relate the findings to the results of a series of in vitro platelet function tests. MATERIALS AND METHODS Subjects: All patients were entered into the study through the voluntary screening program of the Washington University Lipid Research Center. They were classified as type IIa-, IIb-, or IV-HLP on the basis of the results of three consecutive determinationsof serum cholesterol,triglyceridesand lipoprotein electrophoresisaccording to the classificationof Beaumont et al (9). The following 95 percentile cutoff points establishedby the Lipid Research Clinic prevalence studies were used: type IIa: LDL-cholesterol(C) '200 mg/dl, triglyceride (TG) t200 mg/dl; type IIb: LDL-C >200 mg/dl, TC '250 mg/dl; type IV: LDL-C 4200 mg/dl, TC .250 mg/dl. The control subjects were apparently healthy subjects selected to match the patients with respect to age and sex as closely as possible. A detailed general medical history was taken from all patients and volunteers, with particular emphasis on previous signs and symptoms and/or documentationof premature atherosclerotic disease, HLP, hypertensionand cigarette smoking in the subjects themselves, as well as the patients' first degree relatives. The relevant characteristics of patients and controls are shown in Table 1. All subjects had a negative personal and family history for abnormal bleeding and had a normal fasting serum glucose concentration. Patients and controlswere on ad libidum diets. None of the patients had been taking lipid lowering or antiplateletdrugs prior to the study. All subjects were instructednot to take aspirin or any medications other than acetaminophenfor 10 dsys prior to the study. Written informed consent was obtained from all patients and controls before they were entered into the study. Procedures: The template bleeding time was determinedby the method of Mielke et al (10). Blood for the in vitro tests was collected from all subjects after an overnight fast by clean venipunctureof an antecubitalvein and two-syringe technique into plastic syringes as follows: 10 ml of blood was collected into a syringe containing no anticoagulant. 2 ml were immediately added to a vacutainer tube containing 3.0 mg of K3-EDTA for determination of hematocrit and blood platelet concentration. Platelets in whole blood or platelet-richplasma (PRP) were counted by a semi-automatedmethod (11) with dailv comuarisonsof the counts with those obtained bv Dhase contrast was determinedby passing the remicroscop;l(12j. Platelet 'IadhesivenessII mainder of the non-anticoaaulatedblood immediatelythrough a segment (13 cm long, internal diameter 0.; mm) of polyvinyl tubing (Tygon, Norton Plastics and Synthetics Div., Akron, Ohio) containing 1.5 g of glass beads (Class N-A, Cataphote Div., Ferro Corp., Cleveland, Ohio) using an infusion pump (transit time 8 set). At the time the first drop of blood emerged from the end of the glass bead column, a stopwatch was started and the blood issuing from the column between 0 and 10, 10 and 20, and 20 and 30 set was collected into separate siliconizedglass tubes containing 0.07 ml of 15% EDTA-solution. The pump was then stopped and the test was immediatelyrepeated using the remainder of the blood in the same syringe and a new glass bead column. The duplicate test was completed in less than 2 min. The platelet concentrationwas
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then determined in each subsample of blood after passage of the column and in an aliquot of blood expressed into an EDTA-tube from the syringe immediately before connection of the syringe with the first glass bead column. The percentage platelet glass bead column retention (plateletIIadhesiveness") was then calculated for each subsample and the results of duplicate tests averaged. 36 ml of blood was collected into a syringe containing4.0 ml of 3.8% trisodium citrate solution. The blood anticoagulantratio was kept constant since all subjects had hematocritsbetween 41 and 46. After gentle mixing, the titrated blood was centrifuged at 180 g for 10 min and the PRP was carefully transferred into a plastic tube using a plastic pipette. The remainder of the blood was centrifugedat 5,000 g for 8 min at 4OC to obtain platelet-poor plasma (PPP). Platelet-freeplasma (PFP) (platelets
RESEARCH 15;95-108 Press Ltd.1979.
Printed
in Great
Britain
oo49-3848/7g/o6ol-oog5
$02.00/o
INCREASED IN VIVO AND IN VITRO PLATELET FUNCTION IN TYPE II- AND TYPE IV-HYPERLIPOPROTEINEIA
J. Heinrich Joist, R. Kendall Baker, and Gustav Schonfeld Departments of Medicine, Preventive Medicine and Pathology, Washington University School of Medicine, St. Louis, Missouri
(Received 29.9.1978; Accepted by
in revised form 12.2.1979. Editor M.A. Packham)
ABSTRACT Platelet function in vivo and in vitro was examined in 10 patients with type IIa-, 8 patients with type IIb-, and 16 patients with type IV-hyperlipidemia (HIP) and 24 control subjects closely matched for age and sex. Patients with type IIb- and IV-HLP showed significantly shorter template bleeding times in the presence of similar blood platelet concentrations. Increased platelet factor 3-availability in intact platelet-rich plasma (PRP) and PRP exposed to collagen was observed in all patient groups. Plasma antiheparin activity was increased in patients with type IIa- a d type IV-HLP. Platelet adhesiveness, platelet aggregation and 1E C-serotonin release in response to ADP, epinephrine and collagen, as well as plasma von Willebrand factor activity, were generally not increased-in either patient group. Increased platelet function in endogenous HLP may be related to both increased platelet turnover secondary to premature atherosclerotic disease and abnormalities of platelet lipid composition induced by HLP-plasma.
INTRODUCTION There is substantial evidence that platelets may play an important role in the development and progression of premature atherosclerotic disease and its complications, such as myocardial infarction and stroke (l-3). A strong association between premature atherosclerotic disease and increased blood lipid levels has also been well recognized (4-6). This evidence and previously
Reprint Requests to: Dr. J.H. Joist, Div. of Hematology-Oncology, St. Louis University School of Medicine, 1325 S. Grand Blvd., St. Louis, MO 63104 Presented in part at the 49th Meeting of the American Heart Association, Miami Beach, Florida, November, 1976.
95
96
PLATELETS:
HYPERLIPOPROTEINEMIA
Vo1.15,No.1/2
reported findings of increased in vitro platelet sensitivityto aggregating and release-inducingstimuli in patients with type II-hyperlipidemia(HLP) (7) raise the question of a possible pathogeneticlink between HLP, increased platelet function and premature atheroscleroticdisease. The present study was undertaken to study the bleeding time as a measure of platelet function in vivo (8) in patients with type II- and IV-HLP and to relate the findings to the results of a series of in vitro platelet function tests. MATERIALS AND METHODS Subjects: All patients were entered into the study through the voluntary screening program of the Washington University Lipid Research Center. They were classified as type IIa-, IIb-, or IV-HLP on the basis of the results of three consecutive determinationsof serum cholesterol,triglyceridesand lipoprotein electrophoresisaccording to the classificationof Beaumont et al (9). The following 95 percentile cutoff points establishedby the Lipid Research Clinic prevalence studies were used: type IIa: LDL-cholesterol(C) '200 mg/dl, triglyceride (TG) t200 mg/dl; type IIb: LDL-C >200 mg/dl, TC '250 mg/dl; type IV: LDL-C 4200 mg/dl, TC .250 mg/dl. The control subjects were apparently healthy subjects selected to match the patients with respect to age and sex as closely as possible. A detailed general medical history was taken from all patients and volunteers, with particular emphasis on previous signs and symptoms and/or documentationof premature atherosclerotic disease, HLP, hypertensionand cigarette smoking in the subjects themselves, as well as the patients' first degree relatives. The relevant characteristics of patients and controls are shown in Table 1. All subjects had a negative personal and family history for abnormal bleeding and had a normal fasting serum glucose concentration. Patients and controlswere on ad libidum diets. None of the patients had been taking lipid lowering or antiplateletdrugs prior to the study. All subjects were instructednot to take aspirin or any medications other than acetaminophenfor 10 dsys prior to the study. Written informed consent was obtained from all patients and controls before they were entered into the study. Procedures: The template bleeding time was determinedby the method of Mielke et al (10). Blood for the in vitro tests was collected from all subjects after an overnight fast by clean venipunctureof an antecubitalvein and two-syringe technique into plastic syringes as follows: 10 ml of blood was collected into a syringe containing no anticoagulant. 2 ml were immediately added to a vacutainer tube containing 3.0 mg of K3-EDTA for determination of hematocrit and blood platelet concentration. Platelets in whole blood or platelet-richplasma (PRP) were counted by a semi-automatedmethod (11) with dailv comuarisonsof the counts with those obtained bv Dhase contrast was determinedby passing the remicroscop;l(12j. Platelet 'IadhesivenessII mainder of the non-anticoaaulatedblood immediatelythrough a segment (13 cm long, internal diameter 0.; mm) of polyvinyl tubing (Tygon, Norton Plastics and Synthetics Div., Akron, Ohio) containing 1.5 g of glass beads (Class N-A, Cataphote Div., Ferro Corp., Cleveland, Ohio) using an infusion pump (transit time 8 set). At the time the first drop of blood emerged from the end of the glass bead column, a stopwatch was started and the blood issuing from the column between 0 and 10, 10 and 20, and 20 and 30 set was collected into separate siliconizedglass tubes containing 0.07 ml of 15% EDTA-solution. The pump was then stopped and the test was immediatelyrepeated using the remainder of the blood in the same syringe and a new glass bead column. The duplicate test was completed in less than 2 min. The platelet concentrationwas
Vo1.15,No.1/2
PLATELETS:
HYPERLIPOPROTEINEMIA
97
98
PLATELETS:
HYPERLIPOPROTEINEMIA
v01.15,N0.1/2
then determined in each subsample of blood after passage of the column and in an aliquot of blood expressed into an EDTA-tube from the syringe immediately before connection of the syringe with the first glass bead column. The percentage platelet glass bead column retention (plateletIIadhesiveness") was then calculated for each subsample and the results of duplicate tests averaged. 36 ml of blood was collected into a syringe containing4.0 ml of 3.8% trisodium citrate solution. The blood anticoagulantratio was kept constant since all subjects had hematocritsbetween 41 and 46. After gentle mixing, the titrated blood was centrifuged at 180 g for 10 min and the PRP was carefully transferred into a plastic tube using a plastic pipette. The remainder of the blood was centrifugedat 5,000 g for 8 min at 4OC to obtain platelet-poor plasma (PPP). Platelet-freeplasma (PFP) (platelets
