Orhan N. Ulutin

A Study on the Pathogenesis of Thrombopathia using the‘Platelet Osmotic‐Resistance Test’

FUNCTIONAL and qualitative platelet disorders in patients whose platelet counts are normal can be classified into three main categories: A. Willebrand-Jirgens type ofthrombopathia. In this disease, the bleeding time is prolonged, and the amount of thromboplastic factor in the platelets (platelet factor 3, phospholipid) is diminished. The prothrombin-consumption test is abnormal, and when the patient’s platelets are used to replace normal platelets in the thromboplastin-generation test a defect is observed. In some cases the platelets vary greatly in size, and giant platelets are encountered. B. GIanxmann-Naegeli-Braunsteiner type of thrombasthenia. In ths type, a clot-retraction defect exists. In some cases the defect can be detected only by thromboelastography. Defective pseudopod formation, lack of spreading of hyalomere and the defect in the aggregation of platelets are seen by electron microscopy (Braunsteiner, Fellinger and Pakesch, 1953; Alatas and Ulutin, 1954). C. Thrombasthenia thrombopathica (Frank, Ulutin and Karaca, 1956). In this type, the defects of the groups A and B are combined. Clot retraction is poor or absent, the platelet defects are seen by electron microscopy and a reduction in the formation of thromboplastin due to the deficiency of thromboplastic factor of platelets is present. Frank (1925, 1956) used the term ‘athrombia’ to describe a syndrome characterized by reduced agglutinability and adhesiveness of platelets combined with a lack of formation of pseudopodia. Patients with this abnormality had a long bleeding time which was thought to be due to a failure of platelet-thrombus formation in the region of the puncture. The condition, athrombia, described by Frank includes cases of the three categories described above. In the cases of primary thrombopathia a defect in the formation of thromboplastin was demonstrated using the prothrombin-consumption test (Bernard and Soulier, 1948; Ekren, 1951) and later with the thromboplastin-generation test (Bernard, Beaumont and Charreyron, 1953; Marx, 1954; Jurgens, 1955; Soulier, Larrieu and Wartelle, 1955; Braunsteiner and Pakesch, 1956). In 1954 we encountered 18 cases of the Willebrand-Jurgens type of thrombopathia, two cases of the Glanzmann-Naegeli-Braunsteiner type of thrombasthenia and three cases of thrombasthenia thrombopathca. In these cases the defect in the formation of thromboplastin (using the test of Biggs and Douglas, 1953) was corrected when normal platelets were substituted for those of the patient, in a test system containing serum and plasma from the patient. If the patient’s platelets replaced normal platelets in the normal system, the formation of thromboplastin became abnormal. These facts show the existence of a deficiency of the thromboplastic factor of platelets. This defect was seen in 18 cases of thrombopathia of Wdlebrand-Jurgens type and three cases of thrombasthenia thrombopathica. None of these patients had any reduction of anthaemophilic globulin (Factor VIII) or Christmas factor (Factor D() in their blood. We have previously observed that after the incubation of the patient’s platelets with distilled water, the defect in the thromboplastin-generation test disappeared (Ulutin and

A Platelet Defect in a Case Of Scurvy.

IT is generally agreed that the bleeding tendency in scurvy is due to a vascular defect, and Heilmeyer and Begemann (1951)~ Stefanini and Dameshek (1955), Sturgis (1955) and Wintrobe (1956) have stated that in scurvy there is a defect in the synthesis of the cement matter of the capillary endothelium and hence an increased capillary permeability. Stefanini and Dameshek (1955) have also mentioned that thrombocytopenia might be encountered in some cases of scurvy but they do not establish a relationship between this finding and the bleeding tendency in these cases. Except for these statements we have not encountered any report in the literature of a secondary thrombopathy in scurvy. Recently, we have reported a new method for investigating the platelets in the WillebrandJiirgais type of thrombopathy (Frank, Ulutin and Karaca, 1956; Ulutin and Karaca, 1956a, 1956b; Ulutin, 1957). In this disease, when the platelets are treated with distilled water, the ‘third platelet factor’ can be demonstrated. In the secondary thrombopathies, however, such as those which accompany liver disease, uraemia and sprue, platelets treated with distilled water do not yield the durd factor. In the case of scurvy now to be described, the same abnormality was demonstrated, and it is believed to be the first case of secondary thronibopathy in scurvy to be reported.

Apo A-I Binding to Platelets Detected by Flow Cytometry

Lipoprotein-platelet interactions are very important in atherosclerosis and thrombosis. Several studies have been carried out on specific binding of various lipoproteins to platelets. But there is considerable disagreement about the details of these binding sites. Although low-density lipoprotein (LDL) receptors of several cells have been studied extensively, there is little datum about high-density lipoprotein (HDL) receptors. Apolipoprotein (apo) A-I may play a major role in the determination of the specificity of HDL receptors. In this study, binding of apo A-I to platelets was investigated by using a flow cytometric method. Citrated blood samples were obtained from five healthy and seven hypercholesterolemic subjects. Apo A-I antibody was incubated with the citrated whole blood before and after activation with ADP or thrombin receptor agonist peptide (TRAP). Then fluorescein isothiocyanate (FITC)-labeled secondary antibodies were added and analyzed on a Becton-Dickinson FACSort flow cytometer. In the hypercholesterolemic group, apo A-I binding to platelets was found to be significantly decreased after activation with TRAP (P<.05), but not after activation with ADP. In the control group, after platelet activation with ADP or TRAP, the apo A-I MFI values were not found to be significantly different from the values of resting platelets (P>.05). In this study, we demonstrated that apo A-I can bind to platelets, and this supports the hypothesis that apo A-I may play a major role in HDL binding to platelets.

QUALITATIVE PLATELET DISORDERS: CLASSIFICATION AND PATHOGENESIS

A group of congenital, familial, and hereditary platelet diseases has been named “qualitative platelet disorders.” These diseases are characterized by various qualitative anomalies of the platelets. They have been the subject of our studies since 1954. This paper deals with our laboratory findings in such cases and our view regarding their pathogenesis and nomenclature, while comparing our findings with those reported in the literature. Our findings on this matter have been reviewed in various a r t i c l e ~ . ~ ~ ~ T Our original classification of primary qualitative platelet diseases comprised two main groups. The first group included the diseases with an isolated anomaly of the platelets (isolated qualitative diseases of the platelets) Thrombopathy, thrombasthenia, thrombasthenic thrombopathy, and congenital athrombia belong to this group. In the second group, a deficiency of the coagulation factors is also present, in addition to platelet defect (associated qualitative platelet diseases). The von Willebrand’s syndrome is part of this group. In my opinion, the recent findings have brought no essential change in the classification of these disorders. The one view that would be worth while to discuss is the notion that thrombopathy is a syndrome that embraces various types. Thus, primary thrombopathy and thrombasthenic thrombopathy are two entities belonging to the thrombopathic syndrome. Furthermore, the understanding of congenital athrombias and the von Willebrand’s syndrome have been classified. I shall now discuss the laboratory findings and pathogenesis of some of the diseases listed in TABLE 1.

Characterization of Platelet Gamma Glutamyltransferase and Its Alteration in Cases of Atherosclerosis

Among the various functional and biochemical alterations in the platelets of cases of atherosclerosis, the membrane alterations occupy an important place. The platelet intrinsic membrane protein gamma glutamyltransferase (GGT), which is involved in glutathione metabolism, has shown decreased activity in cases of atherosclerosis. To add new insights into the pathogenesis of atherosclerosis, GGT is characterized and correlated with other alterations. Triton X-100 solubilized membrane fractions of frozen and thawed platelets of atherosclerotic and normal subjects had 18.66 ± 2.86 mU/109 platelets and 35.67 ± 3.01 mU/109 platelets, respectively. The K m values were the same, 2.08 mmol/L for gamma glutamyl- p-nitroanilide and 5.87 mmol/L for glycylglycine. The V max values were reduced from 100 mU/109 platelets to 41.66 mU/109 platelets for gamma glutamyl-p-nitroanilide and from 45.45 mU/109 platelets to 38.46 mU/109 platelets for glycylglycine. Optimum pH of GGT activity was 8.2, and optimum temperature was 37°C. It had thermal stability with a 64% relative activity at 36°C for 30 min. Serine against borate was detected as the competitive inhibitor and bromcresol green as the noncompetitive inhibitor. In vivo administration of the antithrombotic drug defibrotide increased the platelet GGT levels to those of normals, from 14.72 ± 7.27 mU/109 platelets to 31.80 ± 12.21 mU/109 platelets in 2 hs. Cholesterol, high-density lipoprotein cholesterol in the membrane fractions, and platelet glutathione levels were unaltered. The lipid per-oxidation (membrane malondialdehyde) level was increased, and glucose and histidine active transport systems were impaired in atherosclerotics. All of these changes are discussed in relation to GGT. Key Words: Human platelets—Atherosclerosis—Gamma glutamyltransferase—Gamma glutamyl transpeptidase— Defibrotide—Biological membranes.

Palmitic Acid Transport in Platelets of Normal Subjects and of Patients with Liver Cirrhosis

Bovine serum albumin bound 14C-palmitic acid (BSA-14C-PA) is transported into platelets of normal and cirrhotic subjects by simple diffusion. Initial uptake increases linearly with the concentration of BSA-14C-PA in the medium. The time course accumulation of BSA-14C-PA is found to be higher in the platelets of patients with cirrhosis compared to that of normals. At 50-min incubation, the amount of 14C-PA accumulated in the platelets of cirrhotic subjects is 52.43 +/- 7.40 nmol/10(9) platelets and in the platelets of normal controls it is 22.71 +/- 3.14 nmol/10(9) platelets. The diffusion rate of BSA-14C-PA is also higher in the platelets of cirrhotic patients where the slope of the concentration dependency curve for 10(9) platelets at 30 sec is 56.0 +/- 4.8 X 10(-4) liters. This value is 21.6 +/- 2.4 X 10(-4) liters for normal subjects.

Persistent vacuoles in leukocytes: Familial Jordans anomaly

Multiple persistent vacuoles were seen in the neutrophils, monocytes and eosinophils of a 9 year old boy and his 10 year old sister. The siblings were both asymptomatic. In the bone marrow, the cytoplasmic vacuoles were also present in the promyelocytes, myelocytes and metamyelocytes, but not in the myeloblasts and they tended to be single and large in immature cells. The cytoplasmic vacuoles did not stain with PAS, Sudan Black or Oil Red O; Sudan III positivity of the vacuoles was found only in a very small number of granulocytes. The vacuoles appeared as round and bright bodies with phase contrast microscopy. By electron microscopy, the vacuoles contained material of low electron density and had no surrounding membrane. Granulocyte functions were unimpaired. Muscle biopsy showed normal morphology. This anomalous vacuolization of the leukocytes is consistent with familial Jordans anomaly.