Evaluation of Platelet Aggregation in Disorders of Hemostasis

Symposium on Advances in Hematology

Evaluation of Platelet Aggregation in Disorders of Hemostasis Yale S. Arkel, M.D. *

The interest in disorders of hemostasis caused by qualitative platelet dysfunction has been stimulated by the descriptions of the methods used by Born to study platelet aggregation in 1962. 5 ,7 This has provided us with an in vitro technique to quantitate the aggregation of platelets. The pattern of response to a variety of aggregating agents can be monitored and displayed graphically. It is the purpose of this review to acquaint the practicing clinician with platelet aggregation and with some of its more practical applications. Although inherited disorders of platelet function are relatively rare, and are associated with histories of mild bleeding, they can be responsible for serious bleeding in surgery. The aggregometer has enabled the physician to more definitively investigate the patient for suspected platelet dysfunction. There are several excellent reviews of platelet function and qualitative platelet disorders, to which the interested reader is referred for a broader introduction to the subject. lo • 45, 50, 64, 72,77

The Role of Platelets in Hemostasis The following is a brief summary of the descriptions in references 45, 50, and 77. The platelet participates in hemostasis by forming a plug at the site of vascular injury and promoting the humoral clotting mechanism. 60,77 In its role as the primary and immediate defender of vascular integrity it has three major actions: adhesion, release, and aggregation. 45 , 50, 77 The platelet procoagulant activity (PF a) is stimulated and made available when platelets are aggregated with collagen and adenosine diphosphate (ADP).35,77 This activity increases the rate of clot formation. 29 ,61 The sequence of events following the vascular injury can be summarized as follows: platelets will adhere to the exposed subendothelial vascular components, i.e., collagen, microfibriles, and basement membrane,63,77 The adhesion phase is followed by the release of stored platelet constitu*Director of Hematology, St. Michael's Medical Center; Clinical Assistant Professor of Medi· cine, New Jersey College of Medicine and Dentistry, Newark, New Jersey Supported in part by a donation from the Schering Foundation, Bloomfield, New Jersey

Medical Clinics of North America- Vo!. 60, No. 5, September 1976

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ents. It is released platelet ADP which promotes the aggregation of platelets and the primary arrest of bleeding. 60 , 77 The platelet plug is thus formed, providing the first line of hemostatic defense. The stimulated PF 3 activity catalyzes the generation of thrombin and a fibrin clot, thereby consolidating the platelet plug. 77 Thrombin is,50. 77, in addition, a potent stimulator of platelet release and aggregation,50. 52. 77 thereby the process has some features of a potentiating cycle. 50 It can be seen, therefore, that platelet aggregation is pivotal in the formation of the hemostatic plug. Platelet Aggregation The principle of platelet aggregometry is based on the change in optical density (light transmission) through platelet-rich plasma when platelet aggregation has occurred. 5-7,44 The platelet-rich plasma is placerl in a well, through which a constant light source is directed. By keeping the temperature constant at 37° C, and with regulated stirring of the platelets, the reaction of platelets to specific aggregating agents can be monitored. Aggregation will permit an increase in light transmission through the platelet-rich plasma. A pattern of response can be demonstrated graphically by recording the change in light transmission. Platelet aggregation can be induced by many agents;50. 77 however, four are used in the clinical laboratory as having the most practical value. We will, therefore, limit our discussion and description to aggregation induced by collagen, epinephrine, adenosine diphosphate (ADP), and ristocetin. The theories concerning the mechanism of action of the aggregating agents, although fascinating and extremely important in understanding platelet physiology and their hemostatic mechanisms, are beyond the scope of this introductory review. The reader is referred to the excellent articles mentioned in the previous section. Standard Aggregating Agents COLLAGEN. 45 , 50, 77 As mentioned earlier, collagen is one of the subendothelial components that react in vivo to induce platelet adhesion and eventuates in platelet aggregation. Collagen suspensions are prepared from tendons and can be obtained commercially. A dilution of this suspension is added 00 an aliquot of the patient's platelet-rich plasma. The changes in aggregation noted after the addition of a collagen suspension occurs following a lag period (Fig. 1). It is by the release of ADP from platelets that collagen induces aggregation. 77 Therefore, collageninduced aggregation is an indirect measure of release of ADP from platelets and the ability of the platelets to aggregate in response to ADP. EPINEPHRINE,45, 49, 50, 77 Epinephrine has a direct effect on platelets and induces a primary aggregation response. Although epinephrine can induce aggregation, a second phase of aggregation caused by the release of ADP from platelets is noted. 50 It is evident, therefore, that epinephrine will measure an abnormality in primary response, release, and the

EVALUATION OF PLATELET AGGREGATION IN DISORDERS OF HEMOSTASIS

Figure 1.

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Normal platelet aggregation in response to collagen.

ability of the platelets to respond to ADP. Separate first and second phase aggregation can be seen in Figure 2. ADENOSINE DIPHOSPHATE. 5 , 45, 50, 52, 58, 60, 77 When added to plateletrich plasma, ADP causes direct aggregation with no lag period, and depending upon the concentration of ADP used, one can further delineate a clear second phase of aggregation due to release of platelet

Figure 2.

Normal response to epinephrine.

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Figure 3.

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Normal response to adenosine diphosphate (ADP).

ADP. In Figure 3 note the separate first and and second phase aggregation. RISTOCETIN. 36 • 41. 75 Ristocetin-induced platelet aggregation (RIP A). This antibiotic, no longer used therapeutically because it induces thrombocytopenia, is a potent platelet-aggregating agent. 36 Its pathway of action is distinct from those of the previous agents,41 The importance of ristocetin became apparent when it was noted that most patients with von Willebrand's disease lacked a plasma factor necessary for ristocetininduced platelet aggregation. 36 • 75. 76 However, other disorders can be associated with abnormal ristocetin-induced platelet aggregation and the test is not specific for von Willebrand's disease. 76 This will be discussed in more detail. Figure 4 shows a typical ristocetin-induced platelet aggregation curve with a small initial phase followed by the major response.

Other Tests for Qualitative Platelet Disorders Although we are centering our discussion on platelet aggregation, it would be misleading to create the impression that aggregometry is the only modality in evaluating qualitative platelet disorders. The classification and description, particularly of the inherited disorders, include other platelet studies and parameters of dysfunction. The Ivy bleeding time l 1. 31. 37 is an excellent screening test for qualitative platelet disorders. Its recent modifications have standardized the length and depth of the incision, making this test more reproducible. 48 However, platelet dysfunction can be present with a normal bleeding time. IS. 77

EVALUATION OF PLATELET AGGREGATION IN DISORDERS OF HEMOSTASIS

Figure 4.

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Normal ristocetin-induced platelet aggregation.

The clot retraction8 and platelet factor 3 availability29. 61 tests are less useful in screening but severe in the evaluation of specific disorders. Platelet adhesiveness tests are abnormal in most qualitative disorders. 55 • 82 The platelet adhesiveness tests were initially most valuable in the detection of von Willebrand's disease. 32 • 59. 66 Aggregometry is rapidly becoming available to all practicing physicians and it is the purpose of this article to introduce the uninitiated to some of its ramifications.

DEFECTS OF AGGREGATION INHERITED DISORDERS OF AGGREGATION

For the most part, the classification we will follow is that described in references 64 and 77. Abnormal First Phase Aggregation (Thrombasthenic Type) Glanzmann's thrombasthenia, first described in 1918, is a rare autosomal recessive disorder.24 The patients have variable bleeding history and are noted to have an abnormal bleeding time and clot retraction. Abnormalities of a less constant nature have been described, including decreased platelet factor 3 availability. 64. 77 The thrombasthenic platelets

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Figure 5.

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No response to collagen (A), adenosine diphosphate (B), or epinephrine (C).

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Figure 5 Continued.

do not aggregate to any of the standard aggregating agents except ristocetin. 64 • 76, 77 However, they do adhere to collagen and will release in response to collagen and thrombin. 64 , 77 In a recent report by Cohen, Glaser, and Seligsohn,15 they noted the effect of ADP and ATP on ristocetin-induced platelet aggregation in thrombasthenic platelets. Ristocetin-induced platelet aggregation was inhibited by the addition of ADP to the platelet mixture before or after adding ristocetin. ATP prevented the effect of ADP inhibition of ristocetin-induced platelet aggregation. Cohen and colleagues postulated a competitive inhibiting effect for ADP and ATP effect or possibly a membrane morphologic change induced by ATP, making the ristocetin receptor sites accessible. ATP, they proposed, maintained the shape of the platelet membrane, keeping the receptor sites exposed to ristocetin. Bovine fibrinogen induced aggregation had a similar pattern with thrombasthenic platelets. 15 An entity described as essential athrombia25 which is most probably a variant of thrombasthenia, has identical aggregation patterns as demonstrated by flat responses to ADP, collagen, and epinephrine. These patients have prolonged bleeding times with normal clot retractions and other platelet functions. We recently studied a young woman with a hemorrhagic disorder who had a bleeding time of 15 minutes (normal, less than 8 minutes) with a normal clot retraction and platelet factor 3 availability (Figure 5). This patient demonstrated platelets that were totally unresponsive to the aggregating agents.

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Figure 6. Typical pattern of abnormal release. A, Essentially no response to collagen; B, primary aggregation with no second phase, in response to epinephrine;

EVALUATION OF PLATELET AGGREGATION IN DISORDERS OF HEMOSTASIS

Figure 6 Continued. phase.

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C, primary response to adenosine diphosphate with no second

In summary, the first phase (thrombasthenic) group is characterized in the aggregometer by the inability to aggregate in response to ADP, collagen, and epinephrine.

Release Abnormalities 1o , 64, 71, 74 (Thrombopathy, Thrombocytopathy) The characteristic aggregation abnormality in this large heterogeneous group is due to an inability of platelets to release endogenous nucleotides in response to ADP, epinephrine, and collagen. Therefore, in the aggregometer we see a primary response to ADP and epinephrine with no second phase aggregation. There is no aggregation response to collagen. This is the "aspirin-like" defect64 ,77 and serves as the model for the release abnormalities. The clinical syndromes described with this inherited disorder of ineffective release are quite varied and beyond the scope of this article. 64 In addition to aggregation defects, these patients may have decreased platelet factor 3 availability, prolonged bleeding time, and abnormal platelet adhesiveness. A typical pattern of abnormal release is demonstrated in Figure 6. The patient was a young mother with long-standing easy bruising and epistaxis. Her platelet aggregation studies demonstrate a pattern compatible with the group of release abnormalities.

Storage Pool Disease64 • 77 Another group of disorders in which the aggregation patterns will have the "aspirin-like" picture consists of the storage pool deficiency disorders. 77 It is the deficiency of adenine nucleotides in the platelet storage pool compartment which prevents the second phase of aggregation. A fairly wide variety of clinical syndromes have been reported.

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Table 1.

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Other Inherited Qualitative Platelet Disorders INHERITED DISORDER

CHARACTERISTIC

MAY BE ASSOCIATED WITH THROMBOCYTOPENIA

May-Hegglin anomaly Dominantly inherited with basophilic inclusions in granulocytes, thrombocytopenia and large platelets with few granules

Abnormal collagen-induced aggregation

"Gray platelet syndrome" Platelets reveal a lack of granules with a gray color on Wright-stained blood smears

Variable aggregation abnormalities noted

Macrothrombopathic thrombocytopenia Autosomal dominant trait with platelet counts in the 20-80,000/cu mm range with giant forms. Megakaryocytes are normal. Most common in patients of Mediterranean extraction and may be associated with other congenital abnormalities, i.e., deafness, nephritis

Variable aggregation (some with a release type abnormality)

Usually differentiated from chronic ITP by a positive family history, the absence of antiplatelet antibodies and the presence of abnormal platelets in the peripheral smear. Ultrastructural studies have revealed a decrease in the number of alpha granules in the platelets (See Fig. 7.) Bernard Soulier syndrome A rare autosomal recessive trait, with giant platelets and lymphocytoid appearance. Clinical picture of moderate to severe bleeding

Decreased to absent aggregation with ristocetin. Normal aggregation with ADP, epinephrine, and collagen

MISCELLANEOUS GROUP

Hereditary afibrinogenemia

Abnormal ADP aggregation

Disorders of Connective Tissue Marfans syndrome Osteogenesis imperfecta Ehlers-Danlos syndrome

Abnormal collagen response may be noted, and release type patterns

This list is a composite from references 64, 70 and 77. For details of specific entities the reader is referred to the list of references.

A storage pool deficiency has been described in a family of patients with the disorder, an additional unrelated group,64 in the Wiskott-Aldrich Syndrome,26 which is a sex-linked disorder characterized by immunologic abnormalities, thrombocytopenia, and eCZema, In the WiskottAldrich syndrome the patient's platelets are smaller than normal with ultrastructural abnormalities. 70 Storage pool deficiencies have also been described in the HemanskyPudlach Syndrome (oculocutaneous albinism)30 and congenital thrombocytopenia associated with absent radii. 19 This group of patients may demonstrate a variable hemorrhagic tendency. Abnormalities in platelet adhesion and platelet factor 3 availability tests have been reported in some of the storage pool deficiencies. 77 The aggregometer, alone, cannot differentiate the release and storage pool group. This requires the quantitation of platelet storage

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pool nucleotides utilizing techniques not readily available in the general clinical laboratory. Other inherited qualitative platelet disorders with abnormal platelet function tests have been reported. The aggregation abnormalities reported are listed in Table 1 and are varied, with no consistent patterns. This table represents a compil:'!.tion of several review articles, principally 64, 70, and 77. Figure 7 demonstrates the pattern of a patient with macrothrombopathic thrombocytopenia.

Von Willebrand's Disease In the strictest sense, von Willebrand's disease is not a primary platelet disorder; nevertheless, platelet aggregation studies can be useful in the evaluation of suspected cases. The classic complex of findings in von Willebrand's disease is a prolonged bleeding time, decreased Fac-

Figure 7. Pattern of platelet aggregation in a patient with macrothrombopathic thrombocytopenia. A, Slight primary response to adenosine diphosphate with no second phase; B, flat response to epinephrine; C, decreased response to collagen.

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tor VIII procoagulant activity, Factor VIII antigen, and decreased platelet adhesiveness to glass beads (platelet retention).4. 9. 59. 64.77 Platelet aggregation in response to ADP, collagen, and epinephrine are normal in the classic case. 77 Ristocetin-induced platelet aggregation is abnormal in patients with von Willebrand's disease. 36 ,75,76 Weisss noted that 13 of 18 patients that he studied had abnormal ristocetin-induced platelet aggregation. 76 The five with normal ristocetin-induced platelet aggregation had a mild clinical history. He noted, however, that ristocetin-induced platelet aggregation was abnormal in some of the patients with other platelet defects, but unlike the group with von Willebrand's disease, their abnormal ristocetin-induced platelet aggregation could not be corrected with normal plasma. 76 Ristocetin-induced platelet aggregation in patients with Bernard-Soulier's syndrome was consistently abnormal and was not correctable with normal plasma. The patient with von Willebrand's disease is lacking a factor in his plasma that is distinct from Factor VIII procoagulant activity.36. 75. 76 This factor (von Willebrand's Factor VIII) is closely associated with Factor VIII antigen. Their relationship has not been completely delineated at this time. 77 It is apparent that ristocetin-induced platelet aggregation requires the presence of von Willebrand Factor VIII to exert its aggregation effect on plateletsY' 74. 76 On this basis, an assay for von Willebrand Factor VIII has been devised using normal washed and fixed platelets and the patient's plasma in a ristocetin aggregation test. I. 43.74 A patient with von Willebrand's disease is demonstrated below (see Figure 8) with abnormal ristocetin-induced platelet aggregation corrected with normal plasma. His epinephrine and ADP and collagen platelet aggregation are normal. Variants of the classic von Willebrand's disease are proposed. The "von Willebrand's syndrome" includes patients with abnormalities in the Factor VIII complex associated with an intrinsic platelet defect and/or prolonged bleeding time. 76 . 77 Figure 9 demonstrates the aggregation pattern of a patient with a release type platelet defect who had abnormal ristocetin-induced platelet aggregation and low Factor VIII levels. Table 2 lists the inherited disorders. ACQUIRED DISORDERS OF AGGREGATION

Qualitative platelet abnormalities have been demonstrated in many disorders including uremia,56 myeloproliferative disease,13, 51. 62 acute leukemia,16.65 immune thrombocytopenia,14. 38. 39. 40 pernicious anemia,42 cirrhosis,68 dysproteinemias,57 congenital heart disease,46 and hypo thyroidism,17 postoperatively,53 and secondary to drugs. 2o . 34. 50. 54 A correlation between bleeding and the abnormalities in platelet aggregation noted is not always well established; however, we will describe a few of the above entities in which the aggregometer can detect abnormal platelet function and may provide some aid in differential diagnosis and prognostication.

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Figure 8. Patient with Von Willebrand's disease. A, Abnormal ristocetin-induced platelet aggregation. B, Correction with normal plasma.

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r

Figure 9. Aggregation pattern of a patien't with a release type of platelet defect with abnormal response to ristocetin and low levels of Factor VIII. A, Abnormal second phase with adenosine diphosphate. B, Absent second phase in the response to epinephrine.

EVALUATION OF PLATELET AGGREGATION IN DISORDERS OF HEMOSTASIS

Figure 9 Continued. ristocetin.

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C, Abnormal response to collagen. D, Abnormal response to

Normal

Normal

Absent

Absent

Classic von Willebrand's disease

Bernard-Soulier syndrome

Thrombasthenia

Release abnormalities "Aspirin-like" abnormality Normal first phase Abnormal second phase

Absent

Normal

Normal

ADP

Normal first phase Abnormal second phase

Normal

Normal

Absent

Normal Absent

Decreased

RISTOCETIN

Normal

EPINEPHRINE

May have abnormal PF" and platelet retention

PF" decreased and clot retraction abnormal

Normal Factor VIII

Factor VIII Antigen-Decreased Factor VIII AHF':'-Decreased Abnormal platelet retention

"Procoagulant activity Note: This chart is based on material supplied at the Hemostasis section of the Education program of the Annual Meeting of the American Society of Hematology, December 6-9, 1975, Dallas, Texas. Panel Members: Bowie, EJW, Edgington, TS, Owen, CA Jr. and Weiss, HJ, and reference 77.

Storage pool disease

COLLAGEN

Inherited Disorders Affecting Platelet Aggregation

DISORDER

Table 2.

t"'

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t"'

"' :n > :>:I

>< ;,.

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~

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897

Finally, we will describe changes in platelet aggregation which we have noted in studying normal subjects in a stressful act.

Myeloproliferative Disease Hemorrhagic complications have been well documented in myeloproliferative disease 72 and in recent years, considerable attention has been focused on platelet function 13 • 51, 62, 67 with platelet aggregation abnormalities being reported in several studies. Zucker and Mielke 81 noted that patients with abnormal platelet function and thrombocytosis in myeloproliferative diseases had a high risk of thrombotic and hemorrhagic complications. The groups at risk had a rather consistently abnormal aggregation response to epinephrine. Neemeh and Bowie et a1. 51 demonstrated that epinephrine-induced aggregation was most consistently abnormal in their patients with myeloproliferative disease. They noted an absence of first phase aggregation in cases of polycythemia vera, primary thrombocythemia and agnogenic myeloid metaplasia. The response to ADP and collagen tended to be normal. Tangun G7 reported that 28 of 33 patients with myeloproliferative disease had a parameter of abnormal platelet function, and only 13 of the 28 cases had elevated platelet counts. This demonstrated that the qualitative platelet abnormalities can be present without excessive platelet production. Platelet aggregation abnormalities were noted in 25 of 28 patients. A number of combinations of abnormal aggregation responses were noted. In some of the cases with abnormal aggregation in response to collagen, a striking increase in the lag phase was noted, suggesting a poor adhesion of the myeloproliferative platelets to the collagen fibers. Studies of platelet function before and after treatment (with myelosuppression) did not seem to indicate a consistent improvement. Cardamone et alY studied 21 consecutive patients with myeloproliferative disease for platelet dysfunction. All 21 patients demonstrated some degree of platelet dysfunction which was more severe in those patients with a bleeding diathesis. An abnormal response to epinephrine was the most consistent abnormality. Spaet et a1. 62 studied 3 patients with essential thrombocythemia and noted the absence of a response to epinephrine with a reduced response to ADP, and normal response to collagen. He postulated that the normal response to collagen may explain the normal bleeding time noted in essential thrombocythemia. It would appear at this time that an abnormal response to epinephrine is the most consistent with respect to platelet aggregation in the myeloproliferative diseases. Although ihe exact clinical significance of abnormal platelet aggregation in myeloproliferative disease is not apparent at this time, platelet aggregation may be of help in the evaluation of this group of disorders. A group of patients 23 with elevated platelet counts resulting from secondary thrombocytosis usually had normal platelet aggregation, and when defects were noted they were of a mild degree; abnormal platelet aggregation is a frequent feature of myeloproliferative disease.

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Figure 10. Patient with polycythemia vera. Platelet studies show (A) slight primary aggregation with no second phase in response to adenosine diphosphate; (B) flat response to epinephrine.

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Figure 10 Continued. (C) normal response to collagen.

A patient with documented polycythemia vera was studied when his counts were: hemoglobin 16.3 gm, white blood count 13.0 x 103 per cu mm, platelet count 267,000 per cu mm, hematocrit 51.4 per cent. He had been treated with periodic phlebotomies. Platelet aggregation study revealed a normal response to collagen and abnormal responses to epinephrine and ADP (Fig. 10). ~ A patient with myelofibrosis of long standing demonstrated abnormal responses to collagen and epinephrine and a normal response to ADP (Fig. 11). Acute Leukemia and Pre-Leukemia Sultan and Caen65 studied the platelet function in patients with refractory anemia and increased myeloblasts, sideroblastic anemia, and erythroleukemia. They noted abnormal platelet aggregation in the group of patients studied in whom acute leukemia developed. Platelet function returned to normal with remission. They postulated a stem cell injury responsible for the defect. Whether platelet aggregation will prove to be of significance in evaluating the pre-Ieukemic patient remains to be proven. Cowan and Haut16 carried out platelet function studies, including platelet aggregation tests, on 14 patients with acute leukemia who had not yet had treatment. They noted an absent second wave of ADP-induced and epinephrine-induced aggregation and a marked delay in collagen-induced aggregation. Partial correction of the platelet defects was noted in one patient who entered complete remission. Cowan and Haut postulated that the abnormality appeared to be due to reactivity of the platelet surface and the release reaction.

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Figure 11. A patient with long-standing myelofibrosis demonstrates an abnormal response to epinephrlne CA), no response to collagen CB), and a normal response to adenosine diphosphate CC).

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Figure 11 Continued.

Immune Thrombocytopenic Purpura The presence of a qualitative platelet dysfunction in immune thrombocytopenic purpura has been documented by Clancy.14 In studying platelet function of patients with chronic and intermittent immune thrombocytopenic purpura, he noted that of those with chronic disease, 8 of 8 had abnormal responses to epinephrine, 9 of 10 had abnormal response to collagen, and 8 of 11 had abnormal aggregation response to ADP. The 3 patients with intermittent immune thrombocytopenic purpura had normal platelet aggregation responses. The globulin fraction of the affected patient's serum, when incubated with normal platelet-rich plasma, inhibited ADP-induced aggregation in 10 of 11 patients and collagen-induced aggregation in 7 of 11 patients. The serum of the groups with intermittent disease showed abnormal aggregation response to ADP in 3 of 3 patients and abnormal response to collagen in 2 of 3 patients. Although the more severe form of the disease was associated with abnormal platelet aggregation in this study, a strict correlation with the level of antiplatelet antibody could not be demonstrated. In a more recent study using a lysis~inhibitor assay, the platelet surface antibody was quantitated. 22 The results indicated a predictive value of this method when studying the thrombocytopenic platelets in terms of response to treatment. In a group of 21 consecutive patients with systemic lupus erythematosus, both thrombocytopenic and with normal platelet counts, 12 of 21 had abnormal platelet function with a mixture of abnormal aggregation responses to collagen, epinephrine, and ADP.41a Impairment correlated

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with the severity of the disease. Those with abnormal platelet aggregation responses had higher sedimentation rates and decreased serum complement levels. Antiplatelet activity was noted in the globulin fraction of the sera from those with abnormal responses. The mechanism of action of platelet antibody in disturbing platelet function is not completely understood, but an impairment of the storage or binding of ADP and serotonin to platelet dense granules has been demonstrated. 40 Antiplatelet antibody has been shown to block the uptake of serotonin and enhance the release of radioactive serotonin from normal platelets. 40 An acquired storage pool deficiency has been described with antiplatelet antibody by Zahavi and Marder et al. 79a Treatments with steroids resulted in a return of platelet function to normal with disappearance of anti platelet antibodies. Lackner and Karpatkin,4!b in studying the "easy bruising" syndrome, noted that a significant number of patients with normal platelet counts have antiplatelet antibodies and demonstrate abnormal platelet aggregation. They suggest that thrombopathia of recent origin and without family history may be an autoimmune disorder. The patient with acquired thrombocytopenia could be tested in the clinical laboratory for abnormal platelet aggregation. If present in the clinical setting of immune thrombocytopenic purpura, an anti platelet antibody can be highly suspect. The testing of the patient's serum!4 as described above with normal platelets in the aggregometer may also yield evidence for a platelet antibody. This, of course, is additive information which can help in the more difficult cases of immune thombocytopenic purpura. The predictive value of this information is suggested in the reports described above.

Isoimmune and Drug-Related Antibodies The increased frequency and demand for platelet transfusions in the treatment of patients with depressed bone marrow function makes the more efficient use of donors desirable and necessary. The use of random donors has been associated with alloimmunization and refractoriness to subsequent platelet transfusions. n ,79 Deykin and Hellerstein2 ! demonstrated the use of aggregometry in detecting isoimmune platelet antibodies by demonstrating the lytic effect of isoantibodies on platelets. They also used this technique in demonstrating quinidineinduced platelet antibody. Wu et aP8 demonstrated that the serum of recipients who were in a refractory state after multiple platelet transfusions, induced aggregation of donor platelets. A correlation between platelet aggregation and HL-A -histocompatibility was noted. Still remaining to be answered is the functional clinical effectiveness of unmatched HL-A platelets when matched platelets are not available. However, platelet aggregation may serve to provide a practical screening method to obtain the most desirable platelets in a clinical setting when HL-A typing is not readily available.

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Uremia The bleeding complications and platelet dysfunctions in uremia have been studied in great detail,36.70 Although several qualitative disorders have been described, the presence of a platelet aggregation abnormality is not clear. Ballard and Marcus 3 were able to correct the abnormal aggregation by changing the ionized calcium levels. However, phenolic acid, phenol, and GSA (guanadinosuccinic acid) present in uremic plasma have been demonstrated to inhibit platelet function and aggregation. 56 Further work is required to clarify the changes in platelet aggregation noted in the uremic patient. The platelet dysfunction associated with uremia will usually improve with hemodialysis or peritoneal dialysis. 56. 70 Patients with cirrhosis have been noted to have altered platelet function. 6s . 7o However, in the cases described, there was also evidence to suggest increased levels of fibrinogen degradation products (FDP) which could have been responsible for the decrease in the aggregation response to ADP and thrombin. Thomas6~ noted that thrombin and plasmin, which may be poorly cleared by the liver, have direct effects on platelets which may eventuate in poor platelet function. The abnormal response to ADP was noted to correlate with the degree of abnormality in thrombin time, suggesting the presence of fibrinogen degradation products. A smaller group of patients had accelerated aggregation responses to ADP; he suggested that the presence of polymerizing fibrin potentiated platelet aggregation. Megaloblastic Anemia Megaloblastic anemia caused by vitamin Bl2 deficiency has been reported to be associated with poor second phase aggregation which is improved with vitamin Bl2 therapy. Levine 42 reported a qualitative disorder in 3 patients with a vitamin Bl2 deficiency. The patients had poor second phase aggregation and this improved after vitamin Bl2 replacement therapy. The initial prolonged bleeding time was greater than the level expected at that degree of thrombocytopenia.

Post-Surgery Bleeding Q'Brien5 ;1 has noted that postsurgical patients have decreased aggregation response to ADP and collagen. He postulated possible exposure of platelets to ADP as a result of tissue trauma during surgery. The relation of these findings to the anesthetic agents used are suggested in view of their known inhibiting effects on platelet aggregation. 69 (See section on drugs.) In the next section we will describe subjects under stress who demonstrated abnormal platelet aggregation. Whether this is a factor in the surgical patient remains to be determined.

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Figure 12. Platelet aggregation appears to be altered by stress; A, normal response to epinephrine before stress; B, abnormal response after stress;

EVALUATION OF PLATELET AGGREGATION IN DISORDERS OF HEMOSTASIS

Figure 12 Continued.

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C, Return to nonnal pattern 7 days after stress.

Platelet Dysfunction and Stress2 , 28 In a study using a group of normal volunteers composed of house staff physicians, we measured platelet aggregation in response to ADP and epinephrine immediately before, immediately after, 24 hours after, and 7 days after their presenting a case before the Mortality Conference of the hospital. In 19 of 22 subjects, a marked decrease in second phase aggregation was noted, particularly in response to epinephrine. Aggregation was noted to be abnormal for 24 hours after the stressful event in 6 of 6. On day 7, aggregation had returned to normal. No consistent change in platelet count or a drop in platelet ADP/ATP content could be detected. Although no definitive conclusion could be reached at this time concerning the mechanism by which these changes occurred, it would appear that platelet aggregation is altered during stress. The necessity of considering the emotional state of the patient when evaluating platelet aggregation is strongly suggested. A typical aggregation response of a subject immediately before the conference (pre-stress), immediately after the conference (post-stress), and the recovery pattern are demonstrated in Figure 12. Platelet and Drugs20 , 34, 50, 54, 69, 73 (Drug-induced platelet aggregation abnormalities) Alteration in platelet function by drugs has been reviewed in several publications. The relationship between drug-altered platelet function

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Figure 13. A typical pattern of aggregation induced by aspirin. A, Poor primary response with no second phase, in response to epinephrine. B, Primary response to adenosine diphos· phate with no second phase.

EVALUATION OF PLATELET AGGREGATION IN DISORDERS OF HEMOSTASIS

Figure 13 Continued.

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C, Flat response to collagen.

and bleeding is not clear. However, in studies of acetylsalicylic acid and surgical bleeding there is some evidence to suggest increased (although no severe) bleeding. 57 This is not a universal finding and other drugs evaluated for increasing surgical bleeding have not revealed a positive definiti ve correlation. 20 Carbenicillin,12 an antibiotic used in severe gram-negative infections, has been reported to induce abnormal platelet aggregation and is associated with bleeding complications. The effect of carbenicillin on platelet aggregation remains for some 12 days after cessation of the drug. The abnormal aggregation pattern noted is of the release type. A similar finding has been noted with high doses of penicillin. 12 The ever growing list of drugs affecting platelet function and aggregation makes the careful history of drug intake vital in an evaluation of a hemostatic failure. It would be wise to withdraw all medications for at least 10 days prior to obtaining a baseline platelet aggregation study. Aspirin,80 as mentioned in a previous section, inhibits the ability of platelets to release. The "aspirin-like" pattern of aggregation is used to describe the abnormal release group. The presence of aspirin in a multitude of proprietary and prescription drugs probably accounts for its being an extremely common cause of abnormal platelet aggregation. A dose of 300 mg acetylsalicylic acid can affect platelet aggregation for some 4 to 7 days.73,77 This will usually not significantly alter hemostasis. However, in patients with underlying abnormal platelet function or a coexisting coagulopathy, a hemorrhagic diathesis may be precipitated or compounded by the effect of aspirin on platelet function. Other anti-inflammatory agents, such as phenylbutazone and indomethacin, affect platelet aggregation in a similar fashion. However, the

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Inducing Abnormal Platelet Aggregation Responses

DRUG

Chloroquine and Hydroxychloroquine Furosemide Nitrofurantoin Glycerol Guaiacolate (cough preparation)

Penicillin, Carbenicillin Penicillin G

ADP

EPl

COLLAGEN

+ +

+

+

+

+

+ +

+

+

Volatile Anesthetics Halothane Methoxyflurane Diethyl ether Cyclopropane Nitrous oxide

+

Nonsteroidal Anti-inflammatory Acetylsalicylic acid (aspirin) Indomethacin Phenylbutazone

+

Tricyclic compounds Chlorpromazine Imipramine Promethazine

+

+ +

+

Alcohol

+

+

+ + +

+

+

+

+ +

+

+= abnormal

Note: Extracted in major part from reference 34.

abnormality is much shorter lived and quickly clears with cessation of the drug. 73 The ever increasing list of drugs affecting platelet function and aggregation makes it advisable to refer to current listings when evaluating a patient with a functional platelet disorder. References 20, 34, 50 and 73 are excellent reviews of drug-induced platelet defects. Let it suffice for us to mention that in addition to those already described, platelet aggregation abnormalities can be seen with cough preparations, anesthetic agents, tranquilizers, ethyl alcohol,33 diuretics, and antihistimines (Table 3). Figure 13 demonstrates a typical aspirininduced aggregation pattern.

CONCLUSION Platelet aggregometry has provided the clinician with a method to evaluate qualitative platelet disorders. This has enabled us to detect the abnormality in hemostasis in a large number of patients with histories of mild bruising or bleeding who previously remained undiagnosed. It is with the inherited disorders of platelet function and more recently with von Willebrand's disease that platelet aggregometry is most valuable in detecting and characterizing the defect. The significance of abnormal platelet function in the acquired disorders remains to be studied and correlated with its associated clinical patterns.

EVALUATION OF PLATELET AGGREGATION IN DISORDERS OF HEMOSTASIS

909

ACKNOWLEDGME!\T

I would like to express my sincere appreciation to Marjorie Voss, Rae Williams, John Capadonna, Christopher Lowther, and Robert Fisher for their assistance in the preparation of this article.

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