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Cyclic platelet dysfunction in IgE-mediated allergy
Cyclic platelet dysfunction IgE-mediated allergy J. S. Gallagher, G. L. Splansky,
in
Ph.D., I. L. Bernstein, M.D., C. A. Maccia, M.D., B.A., and H. I. Glueck, M.D. Cincinnati, Ohio
Diminished platelet aggregation responses to one or more aggregating agents were found in 25 of 32 patients with nasal allergy studied at the peak of the allergy season. Abnormal in-season platelet aggregation induced by epinephrine and collagen was signijicantly improved when repeated out-of-season, while incomplete platelet aggregation induced by adenosine diphosphate (ADP) and thrombin was unchanged. Recombination of an in-season serum factor with autologous, out-of-season normally aggregating platelets caused marked inhibition of platelet aggregation. Mean bleeding times of 20 symptomatic patients were also prolonged during the height of the pollination season. These data suggest that the allergic diathesis is a model for the study of cyclic, nondrug induction of platelet dysfunction.
In these earlier studies, platelet aberrations were reversed when some patients with high levels of specific and total IgE were reinvestigated 3 months beyond their respective allergy seasons. Current experiments were, therefore, designed to evaluate the apparent association of platelet disorders with specific IgE and the reversibility of this phenomenon in patients with allergic rhinitis.
Suppression of second-wave platelet aggregation has been reported in a heterogeneous group of diseases which include oculocutaneous albinism,’ the Wiskott-Aldrich syndrome,2 congenital bleeding disorders3 and in normal persons who ingest aspirin4 Direct and indirect evidences of platelet dysfunction have also been described in atopic and asthmatic patients.jm8 Recent work from this laboratory demonstrated abnormalities of platelet aggregation in 17 of 33 asthmatic patients in whom drug and dietary factors were fully controlled by hospitalization.’ Abnormal responses were significantly greater after epinephrine-, ADP- , collagen-, and thrombin-induced platelet aggregation in patients with immunologically mediated asthma and serum IgE levels >250 U/ml as compared to patients and normal subjects without immunologic factors and serum IgE levels <250 U/ml. Pollen-specific radioallergosorbent (RAST) binding was also significantly increased in patients with abnormal aggregation as compared to normal platelet responders. Impaired secondary release of serotonin and platelet factor 4 generally paralleled abnormal aggregation patterns in asthmatic patients. These results suggested that the allergic state may affect platelet membrane responsiveness to multiple aggregating agents.
MATERIALS AND METHODS Individuals studied Thirty-two patients with nasal pollinosis for two successive grass or ragweed pollination seasons were evaluated. Clinical sensitivity was corroborated by positive allergic potential scores,g nasal examinations during the allergy seasons, markedly positive skin tests by the skin threshold technique, mean specific IgE binding of 5% or greater, and total IgE levels >250 U/ml. All but two of these patients had been treated with pollen immunotherapy, but the cummulative dose range was considerable (0 to 1.3 x lo6 protein nitogen units). Serial platelet aggregation tests were repeated in 25 of the initial 32 patients at least two months beyond the end of the allergy season. Normal subjects studied as a control group were selected on the basis of completely negative personal and family histories of allergy. Drug-induced inhibition of platelet aggregation may persist for as long as 7 days. Therefore, all patients and normal volunteers were restricted from the use of drugs known to cause platelet aberrations for two weeks prior to the testing. This was accomplished by specific instructions about such prescription and nonprescription drugs and providing a list of these drugs to each patient, as previously described.‘j In addition, volunteers were also requested to avoid all other drugs. Monitoring of drug intake was accomplished with detailed drug and symptom diaries. Compliance with these instructions of drug restrictions was uniformly good. Total
From the Immunology (Allergy) and Hematology Divisions, Departments of Internal Medicine and Pathology, University of Cincinnati College of Medicine. Supported in part by Al-12148 and by G.C.R.C. Grant RR 0068. Received for publication Dec. 2, 1977. Accepted for publication July 3, 1978. Reprint requests to: I. Leonard Bernstein, M.D., 231 Bethesda Ave., Cincinnati, OH 45267. 0091-6749178/100229+07$00.70/0
@ 1978 The C. V. Mosby
Co.
Vol. 62, No. 4, pp. 229-235
230 Gallagher et al. drug abstinence during the two-week test period was reported by all but 2 patients, one of whom was on long-term treatment with alpha-methyldopa and the second on therapy with a proprietary combination of spironolactone and hydrochlorothiazide. Since these particular drugs are not thought to affect platelet aggregation, these patients were included in the study.
Assay systems Platelet studies. Platelet aggregation was studied as previously describedY at the symptomatic peak of the allergy season and at least two months after the end of the allergy season when the patients were completely free of symptoms. Platelet-rich plasma (PRP) was prepared by centrifuging 9 ml of whole blood in 1 ml of buffered citrate (final pH 7.2) at 100 x 15 min. Following an initial platelet count in a Model B Coulter counter, the number of platelets was adjusted to 3 X 10”/mm3 by dilution with autologous platelet-poor plasma (PPP) obtained by spinning PRP at 1,000 x g for 20 min. Siliconized glassware, stirring bars, and plastic syringes were used throughout the study. Samples from normal controls and patients were drawn simultaneously, centrifuged, and tested in a dual-channel aggregometer (Chronolog Corp., Broomall, Pa.). PRP, 0.5 ml, was stirred at 1,200 rpm at 37” C in a curvette 8 mm in diameter. The percent transmittance of PRP was recorded as IO and that of PPP as 100. Normal responses to standard aggregating agents were defined as those capable of inducing pen deflection from 10 to maximum transmittance at the sixth minute of aggregation. An abnormal test was defined as percent transmittance 2 standard deviations below the mean normal percent transmittance. Platelet response to the following freshly prepared reagents* was determined: 33 PM epinephrine; 4.3 PM adenosine diphosphate (ADP); 2.3 pg/ml collagen diluted in 1.7 PM acetic acid, and 0.23 U/ml thrombin. These values are expressed as final concentrations in the cuvette. Because epinephrine and collagen aggregation are time-dependent,‘O experiments with these agents are conducted at least 1 hour after blood collection. Ivy bleeding times” were also performed during and after the allergy season. Recombination experiments were performed by incubating in-season serum with out-of-season autologous platelets in a selected subgroup of 3 allergic rhinitis patients. These cases were chosen because their in-season platelet-aggregating patterns demonstrated complete unresponsiveness to two or more aggregating agents and their platelet counts were high enough to be adjusted to 300,000/mm3 after addition of serum. It was necessary to use serum because in-season plasma was not set aside for these tests in the first phase of the investigation. In-season serum was aged for 6 hours at mom temperature before freezing at -20” C for 2 to 6 months. Out-of-season serum was kept at 37” C for 3 hours and then frozen and thawed three times to approximate experimental conditions of in-season serum. Sera pre*Epinephrine was purchasedfrom Parke, Davis & Co., Detroit, Mich., ADP and collagen from Sigma Chemical Co., St. Louis, MO., and thrombin from Ortho Diagnostics, Raritan, N. J.
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CLIN. IMMUNOL. OCTOBER 1978
pared by either method contained no free thrombin. When 0.1 ml of serum was added to 0.1 ml of bovine fibrinogen (200 mg%) and incubated at 37” C for 2 hours, no visible clot was observed. Because serum may contain other residual aggregatory factors, the suitability of substituting this reactant for PPP in recombination experiments was initially tested in a series of experiments with platelets of 4 normal volunteers. Normal PRP was diluted with sufficient serum (0.075 ml to 0.327 ml) to adjust the total platelet count to 3 x 10”/mm3 in a final volume of 0.5 ml. This recombinant mixture was incubated for 20 minutes at room temperature before aggregation with epinephrine, ADP, collagen, or thrombin. Normal aggregation profiles were observed in all instances, indicating that serum neither inhibited nor potentiated aggregation and therefore could be substituted for PPP in future recombination experiments. Recombination experiments were performed by incubating in-season serum with autologous out-of-season platelets. As with normal platelets, the PRP was diluted with sufficient serum (0.075 ml to 0.327 ml) to adjust the total count of platelets to 3 X lO”/mm” in a final volume of 0.5 ml. Patient recombinant mixtures and simultaneous control (normal platelets, normal serum) were incubated at room temperature for 20 minutes before aggregation with the appropriate aggregating agent. Viability of patient and control platelets was determined by performing simple aggregation tests on aliquots of uncombined PRP at the completion of the recombination experiments. Immunologic tests. Specific IgE was evaluated by skin testing and the RAST procedure. Scratch and intracutaneous skin tests with short ragweed and timothy were performed at dilutions of 1: 30 and 1: 1,000 (W/V), respectively. These tests were interpreted according to the criteria in a prior report.y The RAST was performed with short ragweed and timothy pollen allergens coupled to cyanogen bromide -activated methylcellulose discs. After these discs were incubated with patient’s serum and 12”1-anti-IgE, radioactivity bound to the paper discs was measured in a gamma counter. The percentage of lzJI-anti-IgE bound by the allergosorbent serum complex was the quotient of radioactivity bound to the sorbent matrix divided by total amount of radioactivity contained in lzsI-anti-IgE before it was added to the reaction mixture. Total IgE was measured by the radioimmunosorbent (RIST) test. Statistical analysis. An unpaired Student’s t test was used to compare mean platelet aggregation results and mean bleeding times in the patient and control groups. Nonparametric analysis by the Mann-Whitney U test was also performed to determine whether the distribution of results was different in patient and control groups. A paired t test analysis was employed to compare in- and out-of-season platelet results in the 25 patients who completed in- and out-of-season serial studies.
RESULTS During the peak of the allergy season, the mean concentration of serum IgE was 910 U/ml and the
mean allergen-specific RAST binding was 9.6% in 32
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FIG. 1. Platelet aggregation profiles of 32 nasal pollinosis patients performed in-season (-*-e-J, 25 patients repeated out-of-season (-WB-), and 24 normal controls (-1. Each point represents the mean % of light transmission r SE. Statistical probabilities for the fourth minute of aggregation are shown on the graphs. However, all patient in-season aggregation results are significantly different than normal controls from the second through the eighth minutes of aggregation (see text). After stimulation with all four aggregating agents, epinephrine and collagen-induced aggregations improve significantly out-of-season, whereas ADP and thrombin platelet abnormalities persist.
patients. Two months after the end of the allergy season when symptoms had abated, the mean total IgE level was 1,273 U/ml and the mean allergen-specific RAST binding was 9.3% in 25 patients who returned for the second series of tests. Mean daily nasal symptom scores (nasal secretions, stuffiness, and sneezing) during the week preceding platelet testing were used to compare pre- and postseason symptomatic status in the same group of patients. Symptom scores were significantly higher (p < 0.01) during the allergy season. These data confirm the validity of clinical criteria used for selecting the highly allergic test population. Because most of the patients were undergoing immunotherapy, the cumulative dose of allergenic extracts attained prior to platelet testing was compared to the degree and number of platelet aggregation abnormalities. No significant relationships were obtained. Composite plots of patient and control platelet aggregation tests performed in the original group of 32 patients during the symptomatic part of the allergy season are shown in Fig. 1. It is apparent that highly
significant differences by Student’s t test (p < 0.001) exist between allergic patients and normal controls from the second through the eighth minutes of aggregation after epinephrine-, ADP-, and collagen-induced aggregation. Thrombin-induced aggregation was also significantly different at a confidence level of 0.5%. Higher levels of significance were obtained for each of the aggregating agents when the MannWhitney U test was applied with the probabilities of this analysis for epinephrine, collagen, ADP, and thrombin being p < 0.00005, p < 0.0001, p < 0.00003, and p < 0.00007, respectively. Platelet tests were repeated in 25 patients who were available for out-of-season studies (Fig. 1). A paired t test analysis revealed significant improvement in both epinephrine- (p = 0.015) and collagen- (p = 0.019) induced aggregation. Fig. 2, A and B show in- and out-of-season platelet aggregation profiles for a representative patient utilizing epinephrine as the aggregating agent. The in-season response is extremely abnormal as exhibited by the flat profile showing no primary or secondary waves of aggregation, while the
232 Gallagher et al.
TABLE I. Number
J. ALLERGY
of patients
with abnormal
Aggregating agent
Patients with a single study in-season
Epinephrine Collagen ADP Thrombin
12 (32) 11 (30) 16 (32) 10 (30)
results at the sixth minute
of platelet
CLIN. IMMUNOL. OCTOBER 1979
aggregation*
Patients with serial studiest In-season
Out-of-season
10 (25) 9 (25) 13 (25) 7 (25)
6 (25) 5 (25) 11 (24) 8 (23)
Normal subiects
1 (24) 1 (24) 0 (24) 1 (24)
Parenthesesindicate total number of patients tested in each category. *Abnormal platelet aggregation is defined as percent transmittance2 standarddeviations below the mean normal percent transmittance. tSeven patients from the original screening group of 32 were not available for these studies.
out-of-season platelet aggregation pattern is normal with well-characterized primary and secondary waves and maximum aggregation attained at 10 minutes. Although there is a significant improvement between in- and out-of-season mean epinephrine-induced aggregation profiles, the degree of postseasonal reversal of abnormal epinephrine-induced platelet aggregation was not complete. Out-of-season epinephrine-induced platelet aggregation still differed significantly (p = 0.016) from the normal distribution of epinephrine platelet aggregation. On the other hand, in the case of collagen-induced platelet aggregation, the degree of postseasonal reversal of abnormal aggregation was complete since out-of-season mean aggregation to this aggregating agent did not differ significantly from corresponding results in the control population (p = 0.117). Out-of-season ADPand thrombin-induced platelet aggregation profiles did not show improvement in this group of patients. Table I summarizes the number of patients with abnormal platelet aggregation at the sixth minute of the aggregation profile. In the initial series of experiments conducted during the allergy season, 12 patients had abnormal epinephrine-induced aggregation, 11 exhibited abnormal collagen-induced aggregation, 16 had abnormal ADP-induced aggregation, while 10 showed abnormal thrombin-induced aggregation. Normal platelet responses to all four aggregating agents were observed in only seven patients. Thus, 25 of 32 patients demonstrated a total of 49 abnormalities to one or more aggregating agents, as compared to three abnormalities in the control subjects. Eight patients exhibited abnormal platelet aggregation to one aggregating agent, 9 patients reacted abnormally to two aggregators, 7 patients had abnormal responses to three of the platelet stimulators, and 1 patient demonstrated impaired aggregation to all four aggregating substances. Ivy bleeding times were compared between 20 symptomatic patients and 20 asymptomatic controls.
Mean bleeding time of the control population was 3.69 + 0.89 minutes while the patient mean inseason bleeding time was 4.85 + 1.67 minutes. Statistical analysis of these results indicated that the mean in-season bleeding time of allergic patients was significantly longer than that of the control population (Student’s t test:p = 0.02; Mann-Whitney U test: p < 0.004). Out-of-season mean bleeding time of the patient population was 4.07t 1.24 minutes, which did not differ significantly from the control bleeding time. Although the allergic population exhibited a significant increase of bleeding times during the allergy season, as comparea to an asymptomatic control population, only 6 allergic patients demonstrated bleeding time changes in the range of 5.5 to 8 minutes that might be considered clinically relevant. In all of these cases, bleeding times determined out-of-season reverted to control values. Earlier recombination studies had suggested that the plasma of several asthmatic patients with abnormal aggregation partially inhibited the aggregation of normal platelets.6 Several experiments of this type were conducted in a subgroup of 3 allergic rhinitis patients with abnormal responses to two or more aggregating agents by incubating their in-season sera with their autologous out-of-season platelets. Fig. 2 shows a series of these experiments utilizing epinephrine as the aggregating agent. The in-season aggregation was extremely abnormal (Fig. 2, A). There was an out-of season reversal to a normal aggregation profile showing both primary and secondary waves of aggregation (Fig. 2, B). When out-of-season autologous PRP was recombined with in-season serum, there was considerable inhibition of platelet response manifested by the abnormal profile showing no primary or secondary waves of aggregation (Fig. 2, C). A control recombinant sample containing out-of-season serum and out-of-season autologous PRP was not significantly affected (Fig. 2, D). Similar results were obtained in other autologous
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recombinant experiments using collagen, ADP, and thrombin as the aggregating agents. Thus it appears that a serum factor may be partially responsible for in-season platelet abnormalities of epinephrine-, ADP-, thrombin-, and collagen-induced aggregation.
DISCUSSION The association of incomplete platelet aggregation with higher levels of total and specific IgE was first demonstrated in a group of allergic asthmatic patients who were originally part of a heterogeneous asthma population subdivided into allergic and nonallergic categories by history and immunologic findings. In the current investigation, this relationship was retested by studying a homogeneous group of 32 patients with documented pollen sensitivity, pollenspecific IgE, and total IgE >250 U/ml. Most of the patients (25 or 78%) selected by such criteria exhibited one or more abnormalities of platelet aggregation and a majority of these subjects (17 or 53%) reacted abnormally to two or more of the aggregating agents. These results confirm our previous report and clearly indicate that incomplete platelet aggregation is associated with the allergic diathesis. The most interesting observation of this series of investigations concerns the relation of platelet aggregation dysfunction induced by specific aggregating agents to the clinical activity of allergic disease. Abnormal epinephrine- and collagen-induced aggregation improved significantly several months after the
end of the allergy season. The aggregatory response to collagen not only improved significantly, but it could also be interpreted as normal at this time because there was no significant difference between out-of-season patients and normal volunteers. In the case of epinephrine, however, the degree of postseasonal reversal of abnormal platelet aggregation did not quite attain normal values. Persistence of some degree of abnormal epinephrine-induced aggregatiion without regard to season probably accounts for the relative ease of detecting aberrant response to this agent in previous investigations. Although seasonal changes of epinephrine- and collagen-induced aggregation occur only in highly allergic patients with elevated levels of specific and total IgE, such variations are not accompanied by significant decreases in absolute titers of specific and total IgE. It therefore appears likely that platelet dysfunction in allergic patients depends upon a unknown factor which appears only during the allergy season. It is possible that such a substance could produce epinephrine- and collageninduced platelet abnormalities independently, or by activation of reaginic mechanisms. The new observation of season-dependent reversal of abnormal platelet aggregation induced by proaggregatory agents appears to explain several minor clinical inconsistencies in one of our earlier reports.s In that study, three extrinsic asthmatics with elevated levels of serum IgE and RAST binding were found to have relatively normal platelet responsiveness. Re-
234
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sults in two of these patients may now be reconciled with the current investigation because by chance platelet tests were conducted well beyond the allergy season in each of them. Similar to previous studies, abnormal platelet responsiveness in atopic patients was almost completely confined to the release reaction of secondary aggregation. Since experimental evidence suggests that the second phase of aggregation is mediated by beta adrenergic receptors, l2 platelet malfunction may represent another in vitro manifestation of partial beta adrenergic blockade in allergic patients. When this possible relationship was first proposed,‘j there were insufficient data to support the hypothesis of beta adrenergic receptor site regulation by reaginic antibody or the chemical mediators of allergic inflammation. However, the current results clearly demonstrated an association with high concentrations of serum IgE and a seasonal fluctuation of platelet abnormalities, occurrences which suggest that reaginic antibody and/or unknown serologic factors could modulate platelet adrenergic receptor sites during the allergy season. This specific mechanism obviously does not account for platelet unresponsiveness observed by us previously in 7 patients with intrinsic asthma and normal IgE levels.6, s It is conceivable that other nonreaginic environmental factors (e.g., infection) may induce or augument the beta blockade in such patients. Biochemical events of intrinsic platelet prostaglandin synthetic pathwaysI are particularly relevant to the observed second-phase platelet abnormalities of current and other investigations.14 It has been demonstrated that several endoperoxide intermediates (PGG, and PGH,), derived from an interaction between arachidonic acid and prostaglandin synthetase, mediate second-phase aggregation and the release reaction. The endoperoxides are converted to thromboxane A2 by a microsomal thrombaxane synthetase system in platelets. Thromboxane A2 potentiates aggregation and accounts for second-phase aggregation induced by ADP, epinephrine, and collagen. More recently, the discovery of a very potent platelet inhibitory compound, prostacyclin, formed by incubation of an arterial wall enzyme with PGG2 or PGH2, suggests a new homeostatic mechanism for platelet aggregation.i5 According to this hypothesis, reduced second-phase aggregation in atopic patients could be due either to suppression of platelet thromboxane synthetase or to enhanced activation of prostacyclin. Further investigation of these platelet regulatory substances in allergic patients would appear to be particularly worthwhile because platelet interactions with various components of IgE-mediated reactions can be evaluated under controlled conditions. For example, it can be determined whether in vitro combination of
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allergen with cell-bound reaginic antibody could activate the enzymatic conversion of PGG2 and PGH2 to prostacyclin and/or thromboxane synthetase. The observation of significantly longer bleeding times of allergic patients during the allergy season as compared to a control population is noteworthy. Since these tests were reversible when they were repeated out-of-season, it is clear that the sequence of bleeding time prolongation paralleled platelet aggregation dysfunction. It should be noted that bleeding time aberrations were not demonstrated in our previous investigation of platelet function in asthmatic patients. This paradox is not completely understood, but it is possible that subclinical bleeding time abnormalities in the asthmatic group of patients could have been masked by concurrent corticosteroid drugs which diminish capillary permeability. The possible clinical significance of bleeding time prolongation during the allergy season requires furhter investigation because of the occurrence of epistaxis in patients with allergic rhinitis. The presence of circulating inhibitory factor was confirmed in this investigation. While this factor had originally been described in plasma,6 the current studies clearly show that it is also present in serum. Moreover, elicitation of the inhibitory effect in autologous experiments eliminates the possibility that our previous homologous experiments could have been artifacts caused by anti-platelet or HLA antibodies . Although more work is required to elucidate the physicochemical properties of serum inhibitor, it is tempting to speculate about a number of prospects. Several classes of small molecular weight compounds could be responsible for the evanescent suppressive effect. It could be argued that inhibition was caused by high serum drug levels, especially in symptomatic patients. However, this possibility is excluded because unusual efforts were made to control the use of all drugs during the fortnight prior to testing. The only exceptions to the rigid drug exclusion design were 2 patients, one of whom was on long-term maintenance therapy with alpha-methyldopa and the other on a combination of spironolactone and hydrochlorothiazide, respectively. Both of these patients exhibited abnormal platelet patterns during the allergy season and complete reversal of the abnormalities after the allergy season. Presumably, if these drugs played a role in the findings, abnormal aggregation should have occurred on both occasions because they had ingested the same drugs prior to both serial test procedures. Many of the IgE-mediated chemical mediators are low molecular weight substances. Two of these, platelet-activating factor (PAF) l6 and histamine, de-
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serve special comment. The in vitro and in vivo effects of these agents are pro- rather than antiaggregatory. In rabbits, preincubation of PAF with platelets reduces subsequent aggregation induced by PAF, but not other aggregation agents.17 Moreover, since both human PAF and histamine are physically unstable under the conditions of the current experiments, it is unlikely that these substances would persist in the serum. The metabolism of catecholamines might be accelerated in response to the stresses of the allergy seasonI and in-season serum might contain higher concentrations of epinephrine. Preincubation of epinephrine-rich serum with platelets could deactivate specific epinephrine receptors on the platelet membrane and produce a refractory state upon subsequent epinephrine stimulation. I9 However, this “desensitization” effect is thought to be stereospecific and would not account for the inhibitory effects of serum upon collagen-, ADP-, and thrombin-induced platelet aggregation. Among the many proteins which might have suppressor activity, primary attention obviously should be directed to the role of immunoglobulin E. Although human immediate hypersensitivity reactions are thought to be platelet-independent, two groups of investigators have reported specific binding of IgE myeloma protein to human platelet membrane.20*2’ In one of these electron microscopic studies, ferritinconjugated IgE molecules were observed to distribute nonuniformly in clumps or patches on the platelet membrane.21 It is possible that this type of physical rearrangement could interfere with certain enzyme or receptor sites in the membrane. Perturbation of platelet membrane function could be further aggravated by conformational changes of membrane-bound specific IgE after direct interaction with allergen. This would be consistent with the higher incidence of epinephrine- and collagen-induced platelet abnormalities during the allergy season as compared to more normal results several months after the end of the season. In summary, we believe that the allergic diathesis is a model for the study of cyclic, nondrug-induced platelet dysfunction. Although the putative role of platelets in immediate hypersensitivity reactions is an unproved premise, recent experimental reports of platelet-basophil interactions22 and platelet IgE receptors ‘a, 2’ taken together with our results of platelet dy&nction in allergic patients, suggest that further investigation of this hypothesis is warranted. REFERENCES 1. Hardisty. R. M., Mills, D. C. B., and Kestsa-Ard, K.: The platelet defect associated with albinism, Br. J. Haematol. 23:619, 1972.
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2. Grottum, K. A., Hovig, T., Holmsen, H., Abrahamsen, A. F., Jeremic, M., and Seip, M.: Qualitative platelet defects and short platelet survival, Br. J. Haematol. 17:373, 1969. 3. Weiss, H. J.: Platelet physiology and abnormalities of platelet function. I, II, N. Engl. J. Med. 293380, 531, 1975. 4. Weiss, H. J.: Abnormalities in platelet function due to defects in the release reaction, Ann. N. Y. Acad. Sci. 201:161, 1972. 5. Fishel, C. W., and Zwemer, R. J.: Aggregation of platelets in B. perrussis-injected mice and atopically sensitive individuals, Fed. Proc. 29640, 1970. (Abst.) 6. Solinger, A., Bernstein, 1. L., and Glueck, H. I.: The effect of epinephrine on platelet aggregation in normal and atopic subjects, J. ALLERGY CLIN. IMMUNOL. 51:29, 1973. 7. Schwartz, H. J., and Bennett, B.: The differential effect of acetylsalicylic acid on in vitro aggregation of platelets from normal, asthmatic and aspirin-sensitive subjects, Int. Arch. Allergy Appl. Immunol. 45:899, 1973. 8. Coffey, R. G., and Middleton, Jr., E.: Increased adenosine triphosphatase activity in platelets of asthmatic children, Int. Arch. Allergy Appl. Immunol. 48:171. 1975. 9. Maccia, C. A., Gallagher, J. S., Ataman, G., Glueck, H. I., and Bernstein, 1. L.: Platelet thrombopathy in asthmatic patients with elevated immunogobulin E. J. ALLERGY CLIN. IMMUN~L. 59: 101, 1977. 10. Rossi, E. C., and Louis, G.: A time-dependent increase in the responsiveness of platelet-rich plasma to epinephrine, J. Lab. Clin. Med. 85300, 1975. 1 I. Ivy, A. C., Nelson, D., and Buchet, G.: The standardization of certain factors in the cutaneous “venostasis” bleeding time technique, J. Lab. Clin. Med. 26:1812, 1941. 12. Mills, D. C. B., and Roberts, G. C. K.: Effects of adrenaline on human blood platelets, J. Physiol. 193443, 1967. 13. Hamberg, M., Svensson, J., and Samuelson, B.: Thrombaxanes: A new group of biologically active compounds derived from prostaglandin endoperoxides, Proc. Natl. Acad. Sci. U.S.A. 72:2994, 1975. 14. Weiss, H. J., Willis, A. L., Kuhn, G., and Brand, H.: Prostaglandin E, potentiation of platelet aggregation induced by LASS endoperoxide: Absent in storage pool disease, normal after aspirin ingestion, Br. J. Haematol. 32:257, 1976. 15. Moncada, S., Vane, J. R., and Higgs, E. A.: Human arterial and venous tissues generate prostacyclin (prostaglandin X), a potent inhibitor of platelet aggregation, Lancet 1: 18, 1977. 16. Benveniste, J.: Platelet-activating factor, a new mediator of certain anaphylaxis and immune complex deposition from rabbit and human basophils, Nature 249:481, 1974. 17. Henson, P. M.: Activation and desensitization of platelets by platelet-activating factor (PAF) derived from IgE-sensitized basophils. 1. Characteristics of the secretory response, J. Exp. Med. 132:937, 1976. 18. Bernstein. 1. L., and Greenland, R.: Catechol excretion in asthma, Fed. Proc. 32:813, 1973. (Abst.) 19. O’Brien, J. R.: Changes in platelet membranes possibly associated with platelet stickiness, Nature 207: 1057, 1966. 20. Hubscher, T.: Role of the eosinophil in the allergic reactions. I. EDI: An esoinophil-derived inhibitor of histamine release, J. Immunol. 114:1379, 1975. 21. Sullivan. A. L., Grimley, P. M.. and Metzger, H.: Electron microscopic localization of immunoglogulin E on the surface membrane of human basophils, J. Exp. Med. 132: 1403, 1971, 22. Hastie, R., Weiss, L., and Levy, D. A.: An electron microscopic study of the antigen-induced degranulation of human basophilic leukocytes, Fed. Proc. 34:loOO. 1975. (Abst.)
in
Ph.D., I. L. Bernstein, M.D., C. A. Maccia, M.D., B.A., and H. I. Glueck, M.D. Cincinnati, Ohio
Diminished platelet aggregation responses to one or more aggregating agents were found in 25 of 32 patients with nasal allergy studied at the peak of the allergy season. Abnormal in-season platelet aggregation induced by epinephrine and collagen was signijicantly improved when repeated out-of-season, while incomplete platelet aggregation induced by adenosine diphosphate (ADP) and thrombin was unchanged. Recombination of an in-season serum factor with autologous, out-of-season normally aggregating platelets caused marked inhibition of platelet aggregation. Mean bleeding times of 20 symptomatic patients were also prolonged during the height of the pollination season. These data suggest that the allergic diathesis is a model for the study of cyclic, nondrug induction of platelet dysfunction.
In these earlier studies, platelet aberrations were reversed when some patients with high levels of specific and total IgE were reinvestigated 3 months beyond their respective allergy seasons. Current experiments were, therefore, designed to evaluate the apparent association of platelet disorders with specific IgE and the reversibility of this phenomenon in patients with allergic rhinitis.
Suppression of second-wave platelet aggregation has been reported in a heterogeneous group of diseases which include oculocutaneous albinism,’ the Wiskott-Aldrich syndrome,2 congenital bleeding disorders3 and in normal persons who ingest aspirin4 Direct and indirect evidences of platelet dysfunction have also been described in atopic and asthmatic patients.jm8 Recent work from this laboratory demonstrated abnormalities of platelet aggregation in 17 of 33 asthmatic patients in whom drug and dietary factors were fully controlled by hospitalization.’ Abnormal responses were significantly greater after epinephrine-, ADP- , collagen-, and thrombin-induced platelet aggregation in patients with immunologically mediated asthma and serum IgE levels >250 U/ml as compared to patients and normal subjects without immunologic factors and serum IgE levels <250 U/ml. Pollen-specific radioallergosorbent (RAST) binding was also significantly increased in patients with abnormal aggregation as compared to normal platelet responders. Impaired secondary release of serotonin and platelet factor 4 generally paralleled abnormal aggregation patterns in asthmatic patients. These results suggested that the allergic state may affect platelet membrane responsiveness to multiple aggregating agents.
MATERIALS AND METHODS Individuals studied Thirty-two patients with nasal pollinosis for two successive grass or ragweed pollination seasons were evaluated. Clinical sensitivity was corroborated by positive allergic potential scores,g nasal examinations during the allergy seasons, markedly positive skin tests by the skin threshold technique, mean specific IgE binding of 5% or greater, and total IgE levels >250 U/ml. All but two of these patients had been treated with pollen immunotherapy, but the cummulative dose range was considerable (0 to 1.3 x lo6 protein nitogen units). Serial platelet aggregation tests were repeated in 25 of the initial 32 patients at least two months beyond the end of the allergy season. Normal subjects studied as a control group were selected on the basis of completely negative personal and family histories of allergy. Drug-induced inhibition of platelet aggregation may persist for as long as 7 days. Therefore, all patients and normal volunteers were restricted from the use of drugs known to cause platelet aberrations for two weeks prior to the testing. This was accomplished by specific instructions about such prescription and nonprescription drugs and providing a list of these drugs to each patient, as previously described.‘j In addition, volunteers were also requested to avoid all other drugs. Monitoring of drug intake was accomplished with detailed drug and symptom diaries. Compliance with these instructions of drug restrictions was uniformly good. Total
From the Immunology (Allergy) and Hematology Divisions, Departments of Internal Medicine and Pathology, University of Cincinnati College of Medicine. Supported in part by Al-12148 and by G.C.R.C. Grant RR 0068. Received for publication Dec. 2, 1977. Accepted for publication July 3, 1978. Reprint requests to: I. Leonard Bernstein, M.D., 231 Bethesda Ave., Cincinnati, OH 45267. 0091-6749178/100229+07$00.70/0
@ 1978 The C. V. Mosby
Co.
Vol. 62, No. 4, pp. 229-235
230 Gallagher et al. drug abstinence during the two-week test period was reported by all but 2 patients, one of whom was on long-term treatment with alpha-methyldopa and the second on therapy with a proprietary combination of spironolactone and hydrochlorothiazide. Since these particular drugs are not thought to affect platelet aggregation, these patients were included in the study.
Assay systems Platelet studies. Platelet aggregation was studied as previously describedY at the symptomatic peak of the allergy season and at least two months after the end of the allergy season when the patients were completely free of symptoms. Platelet-rich plasma (PRP) was prepared by centrifuging 9 ml of whole blood in 1 ml of buffered citrate (final pH 7.2) at 100 x 15 min. Following an initial platelet count in a Model B Coulter counter, the number of platelets was adjusted to 3 X 10”/mm3 by dilution with autologous platelet-poor plasma (PPP) obtained by spinning PRP at 1,000 x g for 20 min. Siliconized glassware, stirring bars, and plastic syringes were used throughout the study. Samples from normal controls and patients were drawn simultaneously, centrifuged, and tested in a dual-channel aggregometer (Chronolog Corp., Broomall, Pa.). PRP, 0.5 ml, was stirred at 1,200 rpm at 37” C in a curvette 8 mm in diameter. The percent transmittance of PRP was recorded as IO and that of PPP as 100. Normal responses to standard aggregating agents were defined as those capable of inducing pen deflection from 10 to maximum transmittance at the sixth minute of aggregation. An abnormal test was defined as percent transmittance 2 standard deviations below the mean normal percent transmittance. Platelet response to the following freshly prepared reagents* was determined: 33 PM epinephrine; 4.3 PM adenosine diphosphate (ADP); 2.3 pg/ml collagen diluted in 1.7 PM acetic acid, and 0.23 U/ml thrombin. These values are expressed as final concentrations in the cuvette. Because epinephrine and collagen aggregation are time-dependent,‘O experiments with these agents are conducted at least 1 hour after blood collection. Ivy bleeding times” were also performed during and after the allergy season. Recombination experiments were performed by incubating in-season serum with out-of-season autologous platelets in a selected subgroup of 3 allergic rhinitis patients. These cases were chosen because their in-season platelet-aggregating patterns demonstrated complete unresponsiveness to two or more aggregating agents and their platelet counts were high enough to be adjusted to 300,000/mm3 after addition of serum. It was necessary to use serum because in-season plasma was not set aside for these tests in the first phase of the investigation. In-season serum was aged for 6 hours at mom temperature before freezing at -20” C for 2 to 6 months. Out-of-season serum was kept at 37” C for 3 hours and then frozen and thawed three times to approximate experimental conditions of in-season serum. Sera pre*Epinephrine was purchasedfrom Parke, Davis & Co., Detroit, Mich., ADP and collagen from Sigma Chemical Co., St. Louis, MO., and thrombin from Ortho Diagnostics, Raritan, N. J.
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pared by either method contained no free thrombin. When 0.1 ml of serum was added to 0.1 ml of bovine fibrinogen (200 mg%) and incubated at 37” C for 2 hours, no visible clot was observed. Because serum may contain other residual aggregatory factors, the suitability of substituting this reactant for PPP in recombination experiments was initially tested in a series of experiments with platelets of 4 normal volunteers. Normal PRP was diluted with sufficient serum (0.075 ml to 0.327 ml) to adjust the total platelet count to 3 x 10”/mm3 in a final volume of 0.5 ml. This recombinant mixture was incubated for 20 minutes at room temperature before aggregation with epinephrine, ADP, collagen, or thrombin. Normal aggregation profiles were observed in all instances, indicating that serum neither inhibited nor potentiated aggregation and therefore could be substituted for PPP in future recombination experiments. Recombination experiments were performed by incubating in-season serum with autologous out-of-season platelets. As with normal platelets, the PRP was diluted with sufficient serum (0.075 ml to 0.327 ml) to adjust the total count of platelets to 3 X lO”/mm” in a final volume of 0.5 ml. Patient recombinant mixtures and simultaneous control (normal platelets, normal serum) were incubated at room temperature for 20 minutes before aggregation with the appropriate aggregating agent. Viability of patient and control platelets was determined by performing simple aggregation tests on aliquots of uncombined PRP at the completion of the recombination experiments. Immunologic tests. Specific IgE was evaluated by skin testing and the RAST procedure. Scratch and intracutaneous skin tests with short ragweed and timothy were performed at dilutions of 1: 30 and 1: 1,000 (W/V), respectively. These tests were interpreted according to the criteria in a prior report.y The RAST was performed with short ragweed and timothy pollen allergens coupled to cyanogen bromide -activated methylcellulose discs. After these discs were incubated with patient’s serum and 12”1-anti-IgE, radioactivity bound to the paper discs was measured in a gamma counter. The percentage of lzJI-anti-IgE bound by the allergosorbent serum complex was the quotient of radioactivity bound to the sorbent matrix divided by total amount of radioactivity contained in lzsI-anti-IgE before it was added to the reaction mixture. Total IgE was measured by the radioimmunosorbent (RIST) test. Statistical analysis. An unpaired Student’s t test was used to compare mean platelet aggregation results and mean bleeding times in the patient and control groups. Nonparametric analysis by the Mann-Whitney U test was also performed to determine whether the distribution of results was different in patient and control groups. A paired t test analysis was employed to compare in- and out-of-season platelet results in the 25 patients who completed in- and out-of-season serial studies.
RESULTS During the peak of the allergy season, the mean concentration of serum IgE was 910 U/ml and the
mean allergen-specific RAST binding was 9.6% in 32
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FIG. 1. Platelet aggregation profiles of 32 nasal pollinosis patients performed in-season (-*-e-J, 25 patients repeated out-of-season (-WB-), and 24 normal controls (-1. Each point represents the mean % of light transmission r SE. Statistical probabilities for the fourth minute of aggregation are shown on the graphs. However, all patient in-season aggregation results are significantly different than normal controls from the second through the eighth minutes of aggregation (see text). After stimulation with all four aggregating agents, epinephrine and collagen-induced aggregations improve significantly out-of-season, whereas ADP and thrombin platelet abnormalities persist.
patients. Two months after the end of the allergy season when symptoms had abated, the mean total IgE level was 1,273 U/ml and the mean allergen-specific RAST binding was 9.3% in 25 patients who returned for the second series of tests. Mean daily nasal symptom scores (nasal secretions, stuffiness, and sneezing) during the week preceding platelet testing were used to compare pre- and postseason symptomatic status in the same group of patients. Symptom scores were significantly higher (p < 0.01) during the allergy season. These data confirm the validity of clinical criteria used for selecting the highly allergic test population. Because most of the patients were undergoing immunotherapy, the cumulative dose of allergenic extracts attained prior to platelet testing was compared to the degree and number of platelet aggregation abnormalities. No significant relationships were obtained. Composite plots of patient and control platelet aggregation tests performed in the original group of 32 patients during the symptomatic part of the allergy season are shown in Fig. 1. It is apparent that highly
significant differences by Student’s t test (p < 0.001) exist between allergic patients and normal controls from the second through the eighth minutes of aggregation after epinephrine-, ADP-, and collagen-induced aggregation. Thrombin-induced aggregation was also significantly different at a confidence level of 0.5%. Higher levels of significance were obtained for each of the aggregating agents when the MannWhitney U test was applied with the probabilities of this analysis for epinephrine, collagen, ADP, and thrombin being p < 0.00005, p < 0.0001, p < 0.00003, and p < 0.00007, respectively. Platelet tests were repeated in 25 patients who were available for out-of-season studies (Fig. 1). A paired t test analysis revealed significant improvement in both epinephrine- (p = 0.015) and collagen- (p = 0.019) induced aggregation. Fig. 2, A and B show in- and out-of-season platelet aggregation profiles for a representative patient utilizing epinephrine as the aggregating agent. The in-season response is extremely abnormal as exhibited by the flat profile showing no primary or secondary waves of aggregation, while the
232 Gallagher et al.
TABLE I. Number
J. ALLERGY
of patients
with abnormal
Aggregating agent
Patients with a single study in-season
Epinephrine Collagen ADP Thrombin
12 (32) 11 (30) 16 (32) 10 (30)
results at the sixth minute
of platelet
CLIN. IMMUNOL. OCTOBER 1979
aggregation*
Patients with serial studiest In-season
Out-of-season
10 (25) 9 (25) 13 (25) 7 (25)
6 (25) 5 (25) 11 (24) 8 (23)
Normal subiects
1 (24) 1 (24) 0 (24) 1 (24)
Parenthesesindicate total number of patients tested in each category. *Abnormal platelet aggregation is defined as percent transmittance2 standarddeviations below the mean normal percent transmittance. tSeven patients from the original screening group of 32 were not available for these studies.
out-of-season platelet aggregation pattern is normal with well-characterized primary and secondary waves and maximum aggregation attained at 10 minutes. Although there is a significant improvement between in- and out-of-season mean epinephrine-induced aggregation profiles, the degree of postseasonal reversal of abnormal epinephrine-induced platelet aggregation was not complete. Out-of-season epinephrine-induced platelet aggregation still differed significantly (p = 0.016) from the normal distribution of epinephrine platelet aggregation. On the other hand, in the case of collagen-induced platelet aggregation, the degree of postseasonal reversal of abnormal aggregation was complete since out-of-season mean aggregation to this aggregating agent did not differ significantly from corresponding results in the control population (p = 0.117). Out-of-season ADPand thrombin-induced platelet aggregation profiles did not show improvement in this group of patients. Table I summarizes the number of patients with abnormal platelet aggregation at the sixth minute of the aggregation profile. In the initial series of experiments conducted during the allergy season, 12 patients had abnormal epinephrine-induced aggregation, 11 exhibited abnormal collagen-induced aggregation, 16 had abnormal ADP-induced aggregation, while 10 showed abnormal thrombin-induced aggregation. Normal platelet responses to all four aggregating agents were observed in only seven patients. Thus, 25 of 32 patients demonstrated a total of 49 abnormalities to one or more aggregating agents, as compared to three abnormalities in the control subjects. Eight patients exhibited abnormal platelet aggregation to one aggregating agent, 9 patients reacted abnormally to two aggregators, 7 patients had abnormal responses to three of the platelet stimulators, and 1 patient demonstrated impaired aggregation to all four aggregating substances. Ivy bleeding times were compared between 20 symptomatic patients and 20 asymptomatic controls.
Mean bleeding time of the control population was 3.69 + 0.89 minutes while the patient mean inseason bleeding time was 4.85 + 1.67 minutes. Statistical analysis of these results indicated that the mean in-season bleeding time of allergic patients was significantly longer than that of the control population (Student’s t test:p = 0.02; Mann-Whitney U test: p < 0.004). Out-of-season mean bleeding time of the patient population was 4.07t 1.24 minutes, which did not differ significantly from the control bleeding time. Although the allergic population exhibited a significant increase of bleeding times during the allergy season, as comparea to an asymptomatic control population, only 6 allergic patients demonstrated bleeding time changes in the range of 5.5 to 8 minutes that might be considered clinically relevant. In all of these cases, bleeding times determined out-of-season reverted to control values. Earlier recombination studies had suggested that the plasma of several asthmatic patients with abnormal aggregation partially inhibited the aggregation of normal platelets.6 Several experiments of this type were conducted in a subgroup of 3 allergic rhinitis patients with abnormal responses to two or more aggregating agents by incubating their in-season sera with their autologous out-of-season platelets. Fig. 2 shows a series of these experiments utilizing epinephrine as the aggregating agent. The in-season aggregation was extremely abnormal (Fig. 2, A). There was an out-of season reversal to a normal aggregation profile showing both primary and secondary waves of aggregation (Fig. 2, B). When out-of-season autologous PRP was recombined with in-season serum, there was considerable inhibition of platelet response manifested by the abnormal profile showing no primary or secondary waves of aggregation (Fig. 2, C). A control recombinant sample containing out-of-season serum and out-of-season autologous PRP was not significantly affected (Fig. 2, D). Similar results were obtained in other autologous
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EPINEPHRINE 33
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recombinant experiments using collagen, ADP, and thrombin as the aggregating agents. Thus it appears that a serum factor may be partially responsible for in-season platelet abnormalities of epinephrine-, ADP-, thrombin-, and collagen-induced aggregation.
DISCUSSION The association of incomplete platelet aggregation with higher levels of total and specific IgE was first demonstrated in a group of allergic asthmatic patients who were originally part of a heterogeneous asthma population subdivided into allergic and nonallergic categories by history and immunologic findings. In the current investigation, this relationship was retested by studying a homogeneous group of 32 patients with documented pollen sensitivity, pollenspecific IgE, and total IgE >250 U/ml. Most of the patients (25 or 78%) selected by such criteria exhibited one or more abnormalities of platelet aggregation and a majority of these subjects (17 or 53%) reacted abnormally to two or more of the aggregating agents. These results confirm our previous report and clearly indicate that incomplete platelet aggregation is associated with the allergic diathesis. The most interesting observation of this series of investigations concerns the relation of platelet aggregation dysfunction induced by specific aggregating agents to the clinical activity of allergic disease. Abnormal epinephrine- and collagen-induced aggregation improved significantly several months after the
end of the allergy season. The aggregatory response to collagen not only improved significantly, but it could also be interpreted as normal at this time because there was no significant difference between out-of-season patients and normal volunteers. In the case of epinephrine, however, the degree of postseasonal reversal of abnormal platelet aggregation did not quite attain normal values. Persistence of some degree of abnormal epinephrine-induced aggregatiion without regard to season probably accounts for the relative ease of detecting aberrant response to this agent in previous investigations. Although seasonal changes of epinephrine- and collagen-induced aggregation occur only in highly allergic patients with elevated levels of specific and total IgE, such variations are not accompanied by significant decreases in absolute titers of specific and total IgE. It therefore appears likely that platelet dysfunction in allergic patients depends upon a unknown factor which appears only during the allergy season. It is possible that such a substance could produce epinephrine- and collageninduced platelet abnormalities independently, or by activation of reaginic mechanisms. The new observation of season-dependent reversal of abnormal platelet aggregation induced by proaggregatory agents appears to explain several minor clinical inconsistencies in one of our earlier reports.s In that study, three extrinsic asthmatics with elevated levels of serum IgE and RAST binding were found to have relatively normal platelet responsiveness. Re-
234
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et al.
sults in two of these patients may now be reconciled with the current investigation because by chance platelet tests were conducted well beyond the allergy season in each of them. Similar to previous studies, abnormal platelet responsiveness in atopic patients was almost completely confined to the release reaction of secondary aggregation. Since experimental evidence suggests that the second phase of aggregation is mediated by beta adrenergic receptors, l2 platelet malfunction may represent another in vitro manifestation of partial beta adrenergic blockade in allergic patients. When this possible relationship was first proposed,‘j there were insufficient data to support the hypothesis of beta adrenergic receptor site regulation by reaginic antibody or the chemical mediators of allergic inflammation. However, the current results clearly demonstrated an association with high concentrations of serum IgE and a seasonal fluctuation of platelet abnormalities, occurrences which suggest that reaginic antibody and/or unknown serologic factors could modulate platelet adrenergic receptor sites during the allergy season. This specific mechanism obviously does not account for platelet unresponsiveness observed by us previously in 7 patients with intrinsic asthma and normal IgE levels.6, s It is conceivable that other nonreaginic environmental factors (e.g., infection) may induce or augument the beta blockade in such patients. Biochemical events of intrinsic platelet prostaglandin synthetic pathwaysI are particularly relevant to the observed second-phase platelet abnormalities of current and other investigations.14 It has been demonstrated that several endoperoxide intermediates (PGG, and PGH,), derived from an interaction between arachidonic acid and prostaglandin synthetase, mediate second-phase aggregation and the release reaction. The endoperoxides are converted to thromboxane A2 by a microsomal thrombaxane synthetase system in platelets. Thromboxane A2 potentiates aggregation and accounts for second-phase aggregation induced by ADP, epinephrine, and collagen. More recently, the discovery of a very potent platelet inhibitory compound, prostacyclin, formed by incubation of an arterial wall enzyme with PGG2 or PGH2, suggests a new homeostatic mechanism for platelet aggregation.i5 According to this hypothesis, reduced second-phase aggregation in atopic patients could be due either to suppression of platelet thromboxane synthetase or to enhanced activation of prostacyclin. Further investigation of these platelet regulatory substances in allergic patients would appear to be particularly worthwhile because platelet interactions with various components of IgE-mediated reactions can be evaluated under controlled conditions. For example, it can be determined whether in vitro combination of
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allergen with cell-bound reaginic antibody could activate the enzymatic conversion of PGG2 and PGH2 to prostacyclin and/or thromboxane synthetase. The observation of significantly longer bleeding times of allergic patients during the allergy season as compared to a control population is noteworthy. Since these tests were reversible when they were repeated out-of-season, it is clear that the sequence of bleeding time prolongation paralleled platelet aggregation dysfunction. It should be noted that bleeding time aberrations were not demonstrated in our previous investigation of platelet function in asthmatic patients. This paradox is not completely understood, but it is possible that subclinical bleeding time abnormalities in the asthmatic group of patients could have been masked by concurrent corticosteroid drugs which diminish capillary permeability. The possible clinical significance of bleeding time prolongation during the allergy season requires furhter investigation because of the occurrence of epistaxis in patients with allergic rhinitis. The presence of circulating inhibitory factor was confirmed in this investigation. While this factor had originally been described in plasma,6 the current studies clearly show that it is also present in serum. Moreover, elicitation of the inhibitory effect in autologous experiments eliminates the possibility that our previous homologous experiments could have been artifacts caused by anti-platelet or HLA antibodies . Although more work is required to elucidate the physicochemical properties of serum inhibitor, it is tempting to speculate about a number of prospects. Several classes of small molecular weight compounds could be responsible for the evanescent suppressive effect. It could be argued that inhibition was caused by high serum drug levels, especially in symptomatic patients. However, this possibility is excluded because unusual efforts were made to control the use of all drugs during the fortnight prior to testing. The only exceptions to the rigid drug exclusion design were 2 patients, one of whom was on long-term maintenance therapy with alpha-methyldopa and the other on a combination of spironolactone and hydrochlorothiazide, respectively. Both of these patients exhibited abnormal platelet patterns during the allergy season and complete reversal of the abnormalities after the allergy season. Presumably, if these drugs played a role in the findings, abnormal aggregation should have occurred on both occasions because they had ingested the same drugs prior to both serial test procedures. Many of the IgE-mediated chemical mediators are low molecular weight substances. Two of these, platelet-activating factor (PAF) l6 and histamine, de-
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serve special comment. The in vitro and in vivo effects of these agents are pro- rather than antiaggregatory. In rabbits, preincubation of PAF with platelets reduces subsequent aggregation induced by PAF, but not other aggregation agents.17 Moreover, since both human PAF and histamine are physically unstable under the conditions of the current experiments, it is unlikely that these substances would persist in the serum. The metabolism of catecholamines might be accelerated in response to the stresses of the allergy seasonI and in-season serum might contain higher concentrations of epinephrine. Preincubation of epinephrine-rich serum with platelets could deactivate specific epinephrine receptors on the platelet membrane and produce a refractory state upon subsequent epinephrine stimulation. I9 However, this “desensitization” effect is thought to be stereospecific and would not account for the inhibitory effects of serum upon collagen-, ADP-, and thrombin-induced platelet aggregation. Among the many proteins which might have suppressor activity, primary attention obviously should be directed to the role of immunoglobulin E. Although human immediate hypersensitivity reactions are thought to be platelet-independent, two groups of investigators have reported specific binding of IgE myeloma protein to human platelet membrane.20*2’ In one of these electron microscopic studies, ferritinconjugated IgE molecules were observed to distribute nonuniformly in clumps or patches on the platelet membrane.21 It is possible that this type of physical rearrangement could interfere with certain enzyme or receptor sites in the membrane. Perturbation of platelet membrane function could be further aggravated by conformational changes of membrane-bound specific IgE after direct interaction with allergen. This would be consistent with the higher incidence of epinephrine- and collagen-induced platelet abnormalities during the allergy season as compared to more normal results several months after the end of the season. In summary, we believe that the allergic diathesis is a model for the study of cyclic, nondrug-induced platelet dysfunction. Although the putative role of platelets in immediate hypersensitivity reactions is an unproved premise, recent experimental reports of platelet-basophil interactions22 and platelet IgE receptors ‘a, 2’ taken together with our results of platelet dy&nction in allergic patients, suggest that further investigation of this hypothesis is warranted. REFERENCES 1. Hardisty. R. M., Mills, D. C. B., and Kestsa-Ard, K.: The platelet defect associated with albinism, Br. J. Haematol. 23:619, 1972.
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2. Grottum, K. A., Hovig, T., Holmsen, H., Abrahamsen, A. F., Jeremic, M., and Seip, M.: Qualitative platelet defects and short platelet survival, Br. J. Haematol. 17:373, 1969. 3. Weiss, H. J.: Platelet physiology and abnormalities of platelet function. I, II, N. Engl. J. Med. 293380, 531, 1975. 4. Weiss, H. J.: Abnormalities in platelet function due to defects in the release reaction, Ann. N. Y. Acad. Sci. 201:161, 1972. 5. Fishel, C. W., and Zwemer, R. J.: Aggregation of platelets in B. perrussis-injected mice and atopically sensitive individuals, Fed. Proc. 29640, 1970. (Abst.) 6. Solinger, A., Bernstein, 1. L., and Glueck, H. I.: The effect of epinephrine on platelet aggregation in normal and atopic subjects, J. ALLERGY CLIN. IMMUNOL. 51:29, 1973. 7. Schwartz, H. J., and Bennett, B.: The differential effect of acetylsalicylic acid on in vitro aggregation of platelets from normal, asthmatic and aspirin-sensitive subjects, Int. Arch. Allergy Appl. Immunol. 45:899, 1973. 8. Coffey, R. G., and Middleton, Jr., E.: Increased adenosine triphosphatase activity in platelets of asthmatic children, Int. Arch. Allergy Appl. Immunol. 48:171. 1975. 9. Maccia, C. A., Gallagher, J. S., Ataman, G., Glueck, H. I., and Bernstein, 1. L.: Platelet thrombopathy in asthmatic patients with elevated immunogobulin E. J. ALLERGY CLIN. IMMUN~L. 59: 101, 1977. 10. Rossi, E. C., and Louis, G.: A time-dependent increase in the responsiveness of platelet-rich plasma to epinephrine, J. Lab. Clin. Med. 85300, 1975. 1 I. Ivy, A. C., Nelson, D., and Buchet, G.: The standardization of certain factors in the cutaneous “venostasis” bleeding time technique, J. Lab. Clin. Med. 26:1812, 1941. 12. Mills, D. C. B., and Roberts, G. C. K.: Effects of adrenaline on human blood platelets, J. Physiol. 193443, 1967. 13. Hamberg, M., Svensson, J., and Samuelson, B.: Thrombaxanes: A new group of biologically active compounds derived from prostaglandin endoperoxides, Proc. Natl. Acad. Sci. U.S.A. 72:2994, 1975. 14. Weiss, H. J., Willis, A. L., Kuhn, G., and Brand, H.: Prostaglandin E, potentiation of platelet aggregation induced by LASS endoperoxide: Absent in storage pool disease, normal after aspirin ingestion, Br. J. Haematol. 32:257, 1976. 15. Moncada, S., Vane, J. R., and Higgs, E. A.: Human arterial and venous tissues generate prostacyclin (prostaglandin X), a potent inhibitor of platelet aggregation, Lancet 1: 18, 1977. 16. Benveniste, J.: Platelet-activating factor, a new mediator of certain anaphylaxis and immune complex deposition from rabbit and human basophils, Nature 249:481, 1974. 17. Henson, P. M.: Activation and desensitization of platelets by platelet-activating factor (PAF) derived from IgE-sensitized basophils. 1. Characteristics of the secretory response, J. Exp. Med. 132:937, 1976. 18. Bernstein. 1. L., and Greenland, R.: Catechol excretion in asthma, Fed. Proc. 32:813, 1973. (Abst.) 19. O’Brien, J. R.: Changes in platelet membranes possibly associated with platelet stickiness, Nature 207: 1057, 1966. 20. Hubscher, T.: Role of the eosinophil in the allergic reactions. I. EDI: An esoinophil-derived inhibitor of histamine release, J. Immunol. 114:1379, 1975. 21. Sullivan. A. L., Grimley, P. M.. and Metzger, H.: Electron microscopic localization of immunoglogulin E on the surface membrane of human basophils, J. Exp. Med. 132: 1403, 1971, 22. Hastie, R., Weiss, L., and Levy, D. A.: An electron microscopic study of the antigen-induced degranulation of human basophilic leukocytes, Fed. Proc. 34:loOO. 1975. (Abst.)