Acta haemat. 55: 216-223 (1976)
Impairment of Platelet Adhesiveness and Platelet Factor 3 Activity in Cyanotic Congenital Heart Disease M. B hargava, S. K. Sanyal, M. K. T hapar . S. K umar and V. H ooja Department of Pathology, Lady Hardinge Medical College and Hospital, and Department of Paediatrics, Safdarjang Hospital, New Delhi
Key Words. Congenital heart disease • Haemostasis • Platelet adhesiveness • Pla telet factor 3 • Platelet function • Polycythemia Abstract. In 33 children with cyanotic congenital heart disease the platelet func tion has been studied. The most significant changes were reduced platelet adhesive ness to glass and impaired availability of platelet factor 3 in nearly 50°/o of the pa tients. Although clot retraction was poor in 84°/o of them, thrombocytopenia and prolonged bleeding time were not significant features.
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Bleeding diathesis in cyanotic congenital heart disease (CCHD), lead ing to severe and even fatal postoperative haemorrhage has been recog nised for some time. A wide variety of haemostatic abnormalities have been reported to be present preoperatively. Thrombocytopenia [12, 20, 24], defective clot retraction [1, 7, 16], prolonged prothrombin time [15, 16, 18, 24], prolonged partial thromboplastin generation time [1, 5, 17], hypofibrinogenaemia and accelerated fibrinolysis [12, 20] have been not ed. Recently, defective aggregation of platelets to ADP or collagen has been recognised [11, 13, 22]. To our knowledge, in none of the available studies impairment of platelet adhesion or platelet factor 3 activity has been found. The purpose of this communication is to describe these plate let abnormalities in CCHD. Coagulation defects were excluded by appro priate laboratory tests.
Bhargava/S anyal/T hapar/K umar/H ooja
217
Fig. 1. Apparatus for measuring platelet adhesiveness to glass.
Materials and Methods 33 patients of both sexes were evaluated. Their ages varied from 1 to 12 years. The diagnoses included Fallot’s tetralogy, total anomalous pulmonary venous drain age, tricuspid atresia and transposition of great vessels. None of the patients dis played evidence of abnormal bleeding at the time of study. They had not received aspirin, or any other drug known to alter platelet function, for at least 10 days prior to study. None of the patients had evidence of renal or liver disease. For tests of coagulation and platelet function, venous blood was collected in siliconised syringes and 9 vol added to 1 vol of 3.2°/o trisodium citrate. The amount of citrate was adjusted proportionally to the volume of packed red cells (VPRC) in polycythaemic subjects with VPRC above 45°/o, using the formula x = 100- VPRC (patient)/55,
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where x = volume of citrate in ml to which whole blood is added to a final volume of 10 ml. Each patient was studied with an age-matched control. The methodologic difficulties involved in separating platelet-rich plasma (PRP) to study platelet function in thrombocytopenic children with polycythacmia are formidable. Insufficient quantities of PRP obtained, resulted in an unequal number of tests in some instances. Haemoglobin was determined as cyanmethaemoglobin using Drabkin’s solution. VPRC was determined by Wintrobe’s method. Routine platelet counts were per formed with ammonium oxalate as the diluent according to H ardisty and I ngram [9j. Bleeding time was measured by Ivy’s method [4], and clot retraction according to Biggs and M acF arlane [2]. Platelet adhesiveness to glass was tested in vitro by a modified Salzman’s tech nique. The apparatus was assembled at the National Physical Laboratory, Delhi (fig. 1). Blood from a clean venipuncture was allowed to flow through a glass col umn 9 cm long, containing 2.5 g of glass beads of 0.5 mm diameter each, at a con stant pressure of 100 mm Hg for a period of 20 sec in each instance. Platelet aggre gation to ADP was measured according to H ardisty and I ngram [9] and to colla gen, by the method of Z ucker and Borelli [26]. Platelet factor 3 (Pf-3) availability was determined by the method based on kaolin clotting time of dilutions of PRP as described by H ardisty and H utton [10],
218
Bhargava/S anyal/T hapar/K umar/H ooja
Table /. Frequency of platelet defects in cyanotic congenital heart disease
Test
Number of patients in vestigated
Patients showing the defect number %
Platelet count Bleeding time Clot retraction Platelet adhesiveness to glass Platelet aggregation to ADP Collagen Pf-3 activity
30 28 32 28
5 3 27 13
16.6 10.7 84.3 46.4
27 11 24
0 5 12
0 46.0 50.0
Table II. Severity of platelet defects in cyanotic congenital heart disease (mean ± SD) Test
Patients
Controls
P
t
Platelet count, x 103///1 Bleeding time, min Clot retraction, % Platelet adhesiveness, % Platelet aggregation with ADP, sec Collagen, % Pf-3 availability, %
232.0 ±86.0 3.7 ±2.0 25.7 ± 12.8 30.0 ±15.9
368.9 ±84.7 2.3 ±1.3 44.7 ±5.8 39.0±4.8
0.05
0.828 0.64
10.2 ± 5.0 35.0± 19.0 4.7
8.8 ±4.6 39.0 ±8.5 >25
Results
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In the 33 patients studied, haemoglobin below 9 g#/o was seen only in 9, in the remaining it ranged between 13 and 22 g%>. 17 children showed VPRC of 50% and above. The incidence of abnormal haemostasis was 100%>. In 25 of the 33 children, two or more haemostatic abnormalities were detected. Platelet dysfunction was isolated in 11 patients while in 18 it coexisted with coagulation defects. The abnormal haemostatic profile was characterised by impairment of clot retraction, reduced platelet adhe sion to glass and poor availability of Pf-3 (table I). As many as 27 of the 33 patients investigated showed poor syneresis of the clot. Amongst these
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Impairment of Platelet Adhesiveness and Platelet Factor 3 Activity in Cyanotic Congenital Heart Disease M. B hargava, S. K. Sanyal, M. K. T hapar . S. K umar and V. H ooja Department of Pathology, Lady Hardinge Medical College and Hospital, and Department of Paediatrics, Safdarjang Hospital, New Delhi
Key Words. Congenital heart disease • Haemostasis • Platelet adhesiveness • Pla telet factor 3 • Platelet function • Polycythemia Abstract. In 33 children with cyanotic congenital heart disease the platelet func tion has been studied. The most significant changes were reduced platelet adhesive ness to glass and impaired availability of platelet factor 3 in nearly 50°/o of the pa tients. Although clot retraction was poor in 84°/o of them, thrombocytopenia and prolonged bleeding time were not significant features.
Downloaded by: King's College London 137.73.144.138 - 9/26/2018 4:54:56 PM
Bleeding diathesis in cyanotic congenital heart disease (CCHD), lead ing to severe and even fatal postoperative haemorrhage has been recog nised for some time. A wide variety of haemostatic abnormalities have been reported to be present preoperatively. Thrombocytopenia [12, 20, 24], defective clot retraction [1, 7, 16], prolonged prothrombin time [15, 16, 18, 24], prolonged partial thromboplastin generation time [1, 5, 17], hypofibrinogenaemia and accelerated fibrinolysis [12, 20] have been not ed. Recently, defective aggregation of platelets to ADP or collagen has been recognised [11, 13, 22]. To our knowledge, in none of the available studies impairment of platelet adhesion or platelet factor 3 activity has been found. The purpose of this communication is to describe these plate let abnormalities in CCHD. Coagulation defects were excluded by appro priate laboratory tests.
Bhargava/S anyal/T hapar/K umar/H ooja
217
Fig. 1. Apparatus for measuring platelet adhesiveness to glass.
Materials and Methods 33 patients of both sexes were evaluated. Their ages varied from 1 to 12 years. The diagnoses included Fallot’s tetralogy, total anomalous pulmonary venous drain age, tricuspid atresia and transposition of great vessels. None of the patients dis played evidence of abnormal bleeding at the time of study. They had not received aspirin, or any other drug known to alter platelet function, for at least 10 days prior to study. None of the patients had evidence of renal or liver disease. For tests of coagulation and platelet function, venous blood was collected in siliconised syringes and 9 vol added to 1 vol of 3.2°/o trisodium citrate. The amount of citrate was adjusted proportionally to the volume of packed red cells (VPRC) in polycythaemic subjects with VPRC above 45°/o, using the formula x = 100- VPRC (patient)/55,
Downloaded by: King's College London 137.73.144.138 - 9/26/2018 4:54:56 PM
where x = volume of citrate in ml to which whole blood is added to a final volume of 10 ml. Each patient was studied with an age-matched control. The methodologic difficulties involved in separating platelet-rich plasma (PRP) to study platelet function in thrombocytopenic children with polycythacmia are formidable. Insufficient quantities of PRP obtained, resulted in an unequal number of tests in some instances. Haemoglobin was determined as cyanmethaemoglobin using Drabkin’s solution. VPRC was determined by Wintrobe’s method. Routine platelet counts were per formed with ammonium oxalate as the diluent according to H ardisty and I ngram [9j. Bleeding time was measured by Ivy’s method [4], and clot retraction according to Biggs and M acF arlane [2]. Platelet adhesiveness to glass was tested in vitro by a modified Salzman’s tech nique. The apparatus was assembled at the National Physical Laboratory, Delhi (fig. 1). Blood from a clean venipuncture was allowed to flow through a glass col umn 9 cm long, containing 2.5 g of glass beads of 0.5 mm diameter each, at a con stant pressure of 100 mm Hg for a period of 20 sec in each instance. Platelet aggre gation to ADP was measured according to H ardisty and I ngram [9] and to colla gen, by the method of Z ucker and Borelli [26]. Platelet factor 3 (Pf-3) availability was determined by the method based on kaolin clotting time of dilutions of PRP as described by H ardisty and H utton [10],
218
Bhargava/S anyal/T hapar/K umar/H ooja
Table /. Frequency of platelet defects in cyanotic congenital heart disease
Test
Number of patients in vestigated
Patients showing the defect number %
Platelet count Bleeding time Clot retraction Platelet adhesiveness to glass Platelet aggregation to ADP Collagen Pf-3 activity
30 28 32 28
5 3 27 13
16.6 10.7 84.3 46.4
27 11 24
0 5 12
0 46.0 50.0
Table II. Severity of platelet defects in cyanotic congenital heart disease (mean ± SD) Test
Patients
Controls
P
t
Platelet count, x 103///1 Bleeding time, min Clot retraction, % Platelet adhesiveness, % Platelet aggregation with ADP, sec Collagen, % Pf-3 availability, %
232.0 ±86.0 3.7 ±2.0 25.7 ± 12.8 30.0 ±15.9
368.9 ±84.7 2.3 ±1.3 44.7 ±5.8 39.0±4.8
0.05
0.828 0.64
10.2 ± 5.0 35.0± 19.0 4.7
8.8 ±4.6 39.0 ±8.5 >25
Results
Downloaded by: King's College London 137.73.144.138 - 9/26/2018 4:54:56 PM
In the 33 patients studied, haemoglobin below 9 g#/o was seen only in 9, in the remaining it ranged between 13 and 22 g%>. 17 children showed VPRC of 50% and above. The incidence of abnormal haemostasis was 100%>. In 25 of the 33 children, two or more haemostatic abnormalities were detected. Platelet dysfunction was isolated in 11 patients while in 18 it coexisted with coagulation defects. The abnormal haemostatic profile was characterised by impairment of clot retraction, reduced platelet adhe sion to glass and poor availability of Pf-3 (table I). As many as 27 of the 33 patients investigated showed poor syneresis of the clot. Amongst these
219
100 -I
