Abstract
Platelet function was evaluated in 29 patients with familial hyperbetalipoproteinemia; 17 were untreated and 12 were receiving clofibrate (Atromid-S). In comparison with 26 normal subjects, the untreated patients aggregated in response to 1/3 the concentration of adenosine diphosphate (ADP), '/25 the concentration of epinephrine, and '/4 the concentration of collagen. Treatment with clofibrate, 2 g daily, returned the ADP

sensitivity of the platelets to normal and returned epinephrine and collagen sensitivity towards normal, even though it did not alter total cholesterol, low-density-lipoprotein (LDL) cholesterol, or triglyceride concentrations significantly.
Platelet nucleotide release was also 3-to-5 fold increased in untreated type II patients. Clofibrate treatment failed to diminish the elevated platelet nucleotide release.
Incubation of clofibrate at therapeutic concentrations (100-200 ,ug/ml) with normal platelets in vitro decreased their sensitivity to ADP and epinephrine and reduced 14C-serotonin release by these agents. The effect on the release was probably mediated through decreased platelet sensitivity since higher doses of epinephrine could reverse the inhibitory effect of clofibrate.
Clofibrate may decrease the incidence of thrombotic complications of atherosclerosis by altering platelet sensitivity to aggregation.
Additional Indexing Words:
Type II hyperlipoproteinemia Thrombosis Hypercholesterolemia Atherosclerosis FAMILIAL HYPERBETALIPOPROTEINEMIA (type II hyperlipoproteinemia), one of the most common hyperlipidemic syndromes, is characterized by the early occurrence of atherosclerosis and its thrombotic complications.' We have shown that type II patients have increased sensitivity to aggregating agents and increased nucleotide release, suggesting that abnormal platelet function may contribute to the thrombotic complications of the type II syndrome. 2 Since clofibrate has been reported to reduce the incidence of thrombotic sequelae of atherosclerosis,3-5 we investigated the effect of this drug on platelet function in type II patients. In addition, platelet func-tion was evaluated after incubating normal platelets and type II platelets with clofibrate in vitro.
Methods

Patient Selection
Twenty-nine patients were studied. All had positive family histories and more than one lipoprotein electrophoretic pattern characteristic of type II hyperlipoproteinemia. They were all on low cholesterol diets; 17 patients (9 women and 8 men) were untreated and 12 were receiving treatment with 2 g clofibrate/day. Twenty-six normal individuals (12 women and 14 men) were selected from healthy laboratory personnel. They had had no medications for more than 15 days, and had normal plasma concentrations of total and low density lipoprotein cholesterol and of triglycerides.
Platelet Function
Preparation of platelet-rich and platelet-poor plasma.
Venous blood was collected through siliconized needles into plastic syringes. Nine volumes of blood were added to one volume of 3.8% aqueous sodium citrate in plastic tubes.
Samples were centrifuged at 230 for 10 min at lOOg; the resultant platelet-rich plasma (PRP) had a platelet count by Coulter counter of 180,000 to 330,000/,u. The remaining blood was centrifuged at 3,000g for 15 min at 4°. The platelet count of this platelet-poor plasma (PPP) was below 20,000/1. Platelet aggregation was studied at 37°C according to a modification2 of the method of Born.6 The aggregometer Circulation, Volume 50, September 1974 570 was set up so that the percent transmittance of platelet-rich plasma was recorded as 0 and that of platelet-poor plasma as 100. The difference between 0 and the maximal transmittance produced by a stimulus was defined as the amplitude of response to that stimulus. Each aggregating agent was tested to determine the lowest concentration capable of inducing the same maximum degree of aggregation characteristic of that substance; the latter was defined as the full response to that agent2 within the range of platelet counts used; aggregation was independent of platelet concentration.
Aggregating agents. ADP* was stored frozen at a concentration of 5 X 10-4M in 0.1 M sodium phosphate buffer, pH 6.8, containing 0.15 M NaCl. A fresh solution of 1epinephrine (Sigma) (2.5 X 10-3M) in distilled water was prepared daily. Acid-soluble calf skin collagen, at a concentration of 5.5 mg/ml (as determined by measuring the hydroxyproline content and multiplying by 7.1)7, 8 was donated by Dr. Joseph Teller (Worthington Biochemicals).
Platelet nucleotide release was studied by the method of Wolfe and Shulman9 substituting gel-filtered for washed platelets. The nucleotide release of the supernate from perchlorate-precipitated platelets was measured spectrophotometrically at 260 mM.
Platelet 14C-serotonin release was measured according to the method of Jerushalmy and Zucker. 15 Clofibrate incubation with platelet-rich plasma (PRP). One mg of sodium ethyl-p-chlorophenoxyisobutyratet (clofibrate) was dissolved in one ml of distilled water. The effect of clofibrate concentrations of 100 ,g/ml and 200 ,g/ml of PRP was evaluated after incubation at 370 for 10, 20, 30, 60, 90, and 120 min by assays of platelet aggregation and`4C-serotonin platelet release. Addition of equivalent volumes of distilled water alone did not affect platelet function. Both normal and type II platelets were studied.
Gel-filtered platelets. Platelets were separated from plasma proteins by gel filtration using a modification of the method of Tangen et al." Tyrode's buffer,12 without albumin, was used to suspend the platelets. After gel filtration, platelets were incubated with sodium clofibrate at 370 for 30 min. Human purified fibrinogen (Kabi, Stockholm), 2 mg/ml platelets, was added prior to the aggregating agents.
The following concentrations of aggregating agents were used: 8 MM ADP, 25,M epinephrine, 110 ,g/ml collagen, per ml platelets. Gel-filtered platelets were also labeled with C-serotonin and the affect of clofibrate on the release reaction was studied. rection for small sample size, was used to compare the data of the two groups. All results were expressed as the mean ± standard error of the mean (SE\I). The concentration of aggregating agents and the total nucleotide release were found to have a log normal distribution. They were plotted semi-logarithmically and the t-test was determined on the logarithm of the individual values.
Results

Characteristics of the Patient Population
The normal individuals had levels of total and LDL cholesterol and triglycerides consistent with previous studies in normal American populations (table 1). The mean concentrations of total and LDL cholesterol in the plasma of untreated type II patients differed significantly from normal (P < 0.01). When treated with clofibrate, these type II patients did not change their levels of total and LDL cholesterol. There were no significant sex or age differences between the patient and normal groups.
Platelet Aggregation in Type II Patients Before and After Clofibrate Treatment
In comparison with 26 normal subjects, the untreated patients responded to 1/3 the concentration of ADP, /25 the concentration of l-epinephrine, and 1/ the concentration of collagen ( fig. 1 ). The differences between the patient and normal means for each of the aggregating agents were highly significant (P < 0.001). Although this effect appeared in vitro after 30 min, and was maximal at one hour, it was demonstrable in vivo only after 6 weeks of clofibrate therapy. The sensitivity of the platelets to ADP returned to normal after clofibrate therapy. Epinephrine sensitivity decreased 13-fold.
Platelet Release Reaction in Type II Patients Before and After Clofibrate Treatment
The release of platelet nucleotides was studied under conditions well above the minimal concentrations for full aggregation (fig. 2 ). Nucleotide release was 3to-5 fold greater in the untreated type II patients than in the normal individuals (P < 0.01). By contrast to the behavior with aggregating agents, platelets from 'able 1 agents. After incubation of PRP with 200 ,g/ml of clofibrate for 1 hr at 370, the mean release with ADP dropped from 51 to 13 (P < 0.01), and with epinephrine from 65 to 25 (P < 0.05). With collagen no significant differences were obtained (P > 0.1). The release of 14C-serotonin from type II platelets was unchanged after incubation with clofibrate. This finding is in agreement with the results obtained with platelets from clofibrate-treated type II patients. Platelets separated from the bulk of plasma proteins by gel filtration responded to clofibrate as did unwashed platelets.
Characteristics of the Patients

Discussion
Platelets from patients with hyperbetalipoproteinemia are hypersensitive in vitro to aggregating agents, and exhibit increased nucleotide release.2 The data presented above suggest that clofibrate returns the platelet sensitivity of type II patients towards normal although the latter finding has been disputed. 17 In our studies, clofibrate in vitro as well as in vivo was able to decrease aggregation of normal or type II platelets. This inhibition was reversible in vitro when higher concentrations of aggregating agents were used. Nucleotide release by platelets from patients treated with clofibrate was unchanged despite their decreased sensitivity to aggregation. Thus, the action of clofibrate is different from that of nonsteroidal antiinflammatory drugs such as aspirin, which primarily affect the release reaction. Perhaps clofibrate alters the platelet membrane to decrease the sensitivity of receptor sites for aggregating agents. The drug itself in its active form is a fatty acid and may be taken up by the platelets.
Although in normal platelets clofibrate inhibits serotonin release, no decrease in serotonin release was noted after incubation of type II platelets with clofibrate. This difference may occur because a given concentration of aggregating agent represents a much greater stimulus to the sensitive type II platelets than to normal platelets.
These observations may have important implications for the treatment of type II hyperlipoproteinemia. Since the platelet abnormality appears to be independent of the plasma cholesterol and LDL concentration and the effect of clofibrate in platelets independent of its effect on lipids, it may be worthwhile to treat all type II patients with clofibrate. It is noteworthy that the drug appears in epidemiologic studies to have a beneficial effect which is independent of any effect on plasma lipids.3 It appears as well to have a distinct effect on easily noted sequelae of atherosclerosis, such as transient cerebral ischemia. 8 Table 3 Effect of Incubation with Clofibrate on 14C-Serotonin Release ADP
Epinephrine
Collagen Normal (4 aM) ( 
57.3
Since clofibrate appears to alter platelet sensitivity to aggregation and aspirin abolishes the release reaction, the combination of the two drugs might be expected to have a greater beneficial effect on intravascular thrombosis than either drug alone. In our limited clinical experience, this appears to be the case; a larger clinical trial of such therapy is now being conducted.

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