Thrombin Formation and Yield in Shed Blood in Relation to Thromboplastins and Prothromboplastins

Abstract

The experiments to be described were undertaken to provide an understanding of the defect in haemorrhagic tendencies connected with the thromboplastin complex. Tn severe haemorrhagic conditions, due to a deficiency of a clotting component, laboratory findings usually correlate well with elinical symptoms, but in a few cases it is difficult to decide whether the haemorrhages are due to a deficiency of a clotting component or to other causes. This is particularly noticeable in suspected thromboplastin deficiencies, because of the lack of techniques which give quantitative results. Blood contains at least two plasma precursors of thromboplastin; anti-haemophilic globulin (Lewis et al., 1946;Brinkhous et al., 1951) and plasma thromboplastin component (PTC), also known as Christmas factor (Aggeler et al., 1952;Biggs et al, 1952). The two components have been called a-and ^-prothromboplastin respectively (Fantl and Sawers, 1954). These are converted on certain surfaces to a-and ^-thromboplastin; the latter two, as well as adequate numbers of thromboeytes and calcium ions, form the blood thromboplastin complex. This reacts with the prothrombin system and thrombin is formed. In addition, blood contains factors which inactivate thrombin. The amount of thrombin which is present in clotting blood at a given moment depends therefore on the balance between formation and inactivation. Under these conditions it is obviously not possible to obtain quantitative results. Recently it was shown that phenols have the property of delaying the inactivation of thrombin by plasma (Fantl, 1954). In the presence of phenols it should be possible to assess quantitatively the ability of a system to form thrombin. With this deviee the relationship between thromboplastin activity and rate of formation and yield of thrombin was investigated. EXPERIMENTAL.Human venous blood was obtained by puncture of an arm vein. Arterial blood was aspirated from the femoral artery. In both cases the first few ml. were discarded. Nine I Part of the expenses of this investigation were defrayed by a grant from the National Health and Medical Reaearch Council, Australia.AustraL J. txp. BioL (1954), 32, pp. 853-866.

The Preservation of Coagulation Factors in Human Plasma

“…Prothrombin was assayed by the method of Fantl (1954a). The rate of formation of thrombin was determined according to Fantl (1954b).…”

“…In each case platelets or brain lipids (BeU and Alton, 1954) were added. The assay procedure of Fantl (1954b) was useti.…”

Thrombin Generation in Normal Subjects and Cardiac Patients

“…26 ' 27 The effect of exercise on other clotting factors is known to be more erratic. 20 The increase of AHG and platelets might play a role in the hypercoagulability observed by us with the thrombin generation test in patients with myocardial infarction. The increase in either or both of these clotting components would explain at least partially the absence of protection against the development of hypercoagulability in some of these patients treated with prothrombin-depressing agents, because these agents have no influence on AHG and platelets and their accelerating effect on prothrombin conversion.…”

“…Similar observations have already been reported with a less sensitive thrombin generation technique. 20 The acceleration of coagulation by dilution of the sample is a well established fact; the enhancement of thrombin generation by dilution is a particular example of this general rule. Dilution of plasma with equal amounts of saline increases the thrombin yield of the mixture two-to threefold.…”

Investigation of a Hæmorrhagic Disease Due to Beta‐prothromboplastin Deficiency Complicated by a Specific Inhibitor of Thromboplastin Formation

“…Reaction mixtures consisted of (i) 0'015 millilitre of normal human oxalated plasma, 0'003 millilitre of brain lipids, o· 3 millilitre of 2 % pyrocatechol in " Veronal " buffer pH 7'4, 0·6 millilitre of 0,02SM CaCl 2 and o'ISM NaCl to a total volume of 1·8 millilitres. Testing according toFantl (1954) (open rectangles). (ii) Same as (i) plus o· 005 millilitre of patient's Baserum (solid triangles).…”