The multiple faces of the partial thromboplastin time APTT

I have enjoyed reading the excellent article on the partial thromboplastin time (PTT) by Gilbert White in the 50th anniversary of the report of PTT [1]. As one of those who used to perform the PTT or activated partial thromboplastin time (APTT) tests by using hands and stopwatches, I feel that they were comparable to the present DNA sequencer or ELISA reader as a method of coagulation research. Thirty years ago, when I was working in the laboratory of Oscar Ratnoff in Cleveland, USA, I did millions of APTT-based assays for contact factors [factor (F)XII, factor (F)XI, Fletcher and Fitzgerald factors] in order to purify these proteins from plasma. I will relate here some aspects of APTT that were not touched upon by White.

There are a wide variety of APTT reagents and protocols. Commercial reagents utilize various activators: Celite, kaolin, micronized silica, and ellagic acid. A common property of these agents is that they are negatively charged and of a high molecular mass; certain positively charged substances, such as Polybrene, lysozyme, and β₂-glycoprotein I, can attach to negatively charged surfaces and inhibit contact activation [2]. Although the APTT is prolonged in any of the deficiencies of the clotting factors involved in the intrinsic pathway, Fletcher factor (prekallikrein) deficiency shows a unique feature: prolongation of the incubation time with activators results in normal or near normal APTT [3,4]. The correction of the abnormal APTT with prolonged incubation does not occur in deficiencies of factor (F)VIII, factor (F)IX, FXI, FXII or Fitzgerald factor (high-molecular-weight kininogen). It is also interesting to note that the APTT test using ellagic acid appears to be insensitive to Fletcher factor deficiency [5].

The exact nature of the clot-promoting properties of activators is not completely understood. Contact activation was considered a result of a general surface property, highly dense negative charges, rather than of specific functional groups, such as a sulfate moiety [6]. Exposure of blood to negatively charged surfaces leads to rapid binding of all contact factors to surfaces. Reciprocal activation of FXII and prekallikrein in the presence of high-molecular-weight kininogen is believed to be the predominant mechanism for the initiation of contact activation.

The APTT has been widely used as a screening test for suspected hemostatic abnormalities. What is the sensitivity of APTT as a screening test for mild coagulation factor deficiencies? The sensitivity may depend on the APTT reagents and protocols. To determine the influence of the titer of individual clotting factors in the APTT, standard pooled plasma is serially diluted with plasmas deficient in individual clotting factor, and the APTT of the mixtures is performed using a 1-min incubation time with kaolin [7]. It was found that the APTT is significantly prolonged if FVIII or FIX activity falls below 40%. Mild hemophilia will be detected by this test.

The APTT-based one-stage assay for clotting factor activity is simple and rapid. However, there are some disadvantages inherent in the assay: the results are affected by the presence of active clotting factors or inhibitors such as lupus anticoagulants and heparin. It is said that the two-stage assay gives more reliable results in FVIII assay [8]. The disadvantages of the two-stage assay are that it is time consuming and the reagents are difficult to prepare.

I believe that, in addition to practical roles, APTT has played an important conceptual role in our understanding of blood coagulation in vitro: intrinsic pathway and extrinsic pathway. As subjects with contact factor deficiency except FXI deficiency were found to have no bleeding tendency and the role of tissue factor in hemostasis was firmly established, the distinction between blood coagulation in vitro and in vivo became more evident, and prolonged APTT does not always indicate a bleeding tendency. As someone who has been interested in the contact phase of blood coagulation, I really miss the role of these factors in hemostasis.

Finally, as White noted some memorable achievements in 1953 in Europe and the USA, I would like to add similar events in Asia. The Korean War ended in July 1953, and Cambodia became independent from France in November of that year. Public nationwide television broadcasting started in Japan in the same year.