Platelets as a Source of Serum Acid Nitrophenylphosphatase*

Abstract

Recent interest in serum enzyme activity in pathological conditions has led to renewed curiosity concerning the source of the enzymes present in serum under normal conditions. Since blood platelets contain acid phosphatase (1-8), it seemed possible that they might liberate this enzyme during blood coagulation. This possibility was investigated by comparing the acid phosphatase activity of serum prepared from platelet-rich and platelet-poor plasmas. Preliminary studies suggested that the platelets contribute virtually all of the activity of normal serum against 8-glycerophosphate at acid pH (9). The present investigation was carried out using p-nitrophenylphosphate as substrate. METHODSAcid phosphatase activity was measured using p-nitrophenylphosphate 1 as substrate (10-12). This method gave highly reproducible values; for example, nine out of 10 determinations on a sample of fresh serum were 0.39 FM per ml. per hour and the tenth was 0.41.The subjects were either healthy young adults or, in a few instances, patients with metastatic carcinoma of the prostate.2 To avoid the use of anticoagulants, native platelet-rich and platelet-poor plasmas were prepared by centrifugation of blood drawn in chilled siliconed syringes and cooled in an ice bath in siliconed tubes. Most of the red and white cells were sedimented by centrifuging for five to 10 minutes at about 1,000 rpm. Two ml. of the supernatant platelet-rich plasma was set aside in an ice bath and a platelet count taken. The remainder of the native blood and platelet-rich plasma was centrifuged in a multispeed attachment at about 10,000 rpm for 10 minutes to obtain platelet-poor plasma. About removed with an applicator stick at the end of incubation, and acid phosphatase activity was measured. These samples are designated as "serum from platelet-rich plasma" and "serum from platelet-poor plasma." The time course of acid phosphatase liberation was studied by placing 0.4 ml. of native platelet-rich plasma in each of a series of new glass tubes (rinsed with distilled water and oven-dried) at 370 C. and, at intervals, removing the platelets. In the samples which had not yet clotted, this was accomplished by brief high-speed centrifugation and the fibrin which formed subsequently in the platelet-poor plasma was removed with an applicator stick. In the samples which had clotted, the clot and entrapped platelets were removed together.To determine whether anticoagulants inhibited enzyme activity, one-fifth volume of 0.1 M sodium oxalate, 0.11 M sodium citrate, or 0.027 M (1 per cent) disodium ethylenediaminetetraacetate in saline (EDTA) was added to serum separated from native platelet-rich plasma or from whole blood. To study the effect of the anticoagulants on the color of nitrophenol, saline was used instead of serum. The activity of whole platelets was tested by comparing the acid phosphatase activity of platelet-rich and platelet-poor plasmas separated from blood in which clotting was prevented by the addition of one-ninth volume of anticoagulant. Other studies we...

Platelet Adhesion in Vivo in Patients with Bleeding Disorders

“…been measured by exposing the platelets to glass under standardized conditions (Wright 1941, Moolten & Vroman 1949, Marx & Derlath 1957, Hellem 1960, and platelet aggregation has been studied by observing plasma under various conditions (Bergsagel 1956, Sharp 1958, and Waaler 1959. In uiuo, similar phenomena have been studied by Bizzozero (1 882), Eberth & Schimmelbusch (1886) and more recently by Zucker (1947) and Hugues ( 1953).…”

A Study of Allograft Kidney Rejection Occurring Simultaneously in Whole Organ and Ear Chamber Grafts in the Rabbit

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Nitrocatecholsulfatase in Human Blood Platelets

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