Intermediate Stages in the Formation of Blood Thromboplastin

RECENTLY it has been demonstrated that an activator of prothrombin is formed by the interaction of calcium, platelets and several plasma factors (Biggs, Douglas and Macfarlane, 1953a). The final activator of prothrombin formed in an incubation mixture of blood constituents is referred to as ‘blood thromboplastin’. The number of coagulation factors required for the formation of blood thromboplastin has not been established, but it is clear that calcium, a platelet factor, antihaemophilic globulin (AHG) and the Christmas factor (C.F.) (Biggs et al., 1952; Aggeler et al., 1952) are essential components. Since it is unlikely that all of the essential factors react simultaneously, Biggs, Douglas and Macfarlane (1953b) suggested that blood thromboplastin is the end-product of a series of intermediate reactions. These authors demonstrated that a preliminary reaction occurs when calcium, AHG and normal serum are incubated together. For convenience, the intermediate product of thromboplastin formed in this reaction will be referred to as Product I. However, we wish to stress that this term is used for convenience only, for it has not been shown that this intermediate product of thromboplastin is the first one to be formed or that only one reaction proceeds when calcium and AHG are incubated with serum. Further studies of the Product I reaction suggest that calcium and a factor (or factors) in serum react stoichiometrically; AHG activity disappears during the formation of Product I and variations in the concentration of AHG have a greater effect on the speed of the reaction than on the amount of Product I formed (Bergsagel and Biggs, 1955). Since it appears that calcium reacts stoichiometrically in the formation of Product I and that Product I is inactivated by decalcifying agents, it has been suggested that this intermediate product of thromboplastin formation is a calciumcomplex (Bergsagel, 1955a, b). Subsequent stages in the formation of blood thromboplastin have not been investigated. Platelets supply an essential component of blood thromboplastin (Brinkhous, 1947; Quick, 1947; Biggs, Douglas and Macfarlane, 1953a), but since an extract of disrupted platelets possesses very little thromboplastic activity (Ware, Fahey and Seegers, 1948) it is clear that this platelet factor reacts with a plasma component before thromboplastic activity develops. The present investigations were undertaken to identify the plasma component which reacts with platelets and to study the properties of the coagulant formed by the reaction. The examination of living platelets by phase-contrast microscopy has shown a cluster of granules (granulomere) surrounded by a clear area (hyalomere). Fonio (1951) separated the hyalomere and granulomere by differential centrifugation of platelets that had been disrupted by suprasonic vibration. The hyalomere, which appears to be responsible for the clotretracting property of platelets, is sedimented by a centrifugal force that leaves the granules in the supernatant. The platelet thromboplastin factor is associated with the granules which are sedimented by a relative centrifugal force of 32,000 g for 90 minutes. t Medical Research Fellow, National Research Council, Canada. * Present address: D.E. B. Department of Medicine, University of Texas M.D. Anderson Hospital and Tumor Institute, Houston 25, Texas, U.S.A. C.H. Department of Pathology, School of Medicine, University of North Carolina, Chapel Hill, N.C. , U.S.A. In receipt of a grant from the Dan Mason Research Foundation.