The Effect of the Proteinase from Rat Peritoneal Mast Cells on Prothrombin and Factor X

Mast cells are usually located perivascularly and the granules contain an ionic complex of heparin, histamine and at least one proteinase. Heparin accelerates the reaction between thrombin and anti-thrombin 111, whereas histamine is a vasodilator and increases capillary permeability. The risk of clotting in the early stages of the inflammatory response to injury is thereby decreased. Although the dissociation of the heparin-proteinase complex in vitro requires high ionic strength, and the mechanism of dissociation in uivo is unknown, it is possible that the proteinase(s) of mast cells or other granulocytes such as basophils may assist in regulating blood coagulation. Accordingly, we have examined the action of the proteinase from rat peritoneal mast cells on prothrombin and Factor X. Prothrombin and Factor X were isolated from bovine and human plasma by the methods of Esnoufet al. (1973) and Bajaj & Mann (1973) in high purity (299 %)and with high specific activities. Mast cells were isolated from rat peritoneal washings by density-gradient centrifugation (20min at 330g) on layers of 30,40 and 50 7; (w/v) Ficoll and were up to 98 % pure. Mast-cell granules were obtained by mild sonication, and the proteinase was differentially extracted between 0.5 and 1 .2~-NacI . The proteinase, which was purified by gel filtration on Sephadex G-100 in 1 .2~-NaCl and was then homogeneous by gel electrophoresis in the presence of 0.1 % sodium dodecyl sulphate, was assayed with 4-methylumbelliferyl p-(NNN-trimethy1ammonium)cinnamate (Jameson et al., 1973). Prothrombin and Factor X were hydrolysed with the mast-cell proteinase and residual clotting activity was assayed (Fig. 1). The rate of inactivation was similar in each case ALASTAIR R. G. WYLIE, JOHN D. LONSDALE-ECCLES,