Blood Platelets and Proteolytic Enzymes

This thesis gives an account of the investigation of the effect of a variety of proteolytic enzymes on platelet aggregation. The discovery of platelets in blood and the recognition of their importance in haemostasis and thrombus formation is described in an historical introduction, which is followed by an account of materials and methods used, including three in vitro tests designed to estimate the ability of platelets to aggregate. Techniques for the preparation, purification and characterization of degradation products formed by proteolytic digestion of fibrinogen are also described. The experimental section of the thesis begins with an account of the assessment in vitro of the effect of fibrinogenolytic agents, particularly streptokinase and trypsin, and of products of fibrinogenolytic activity on platelet aggregation and adhesion. Platelet aggregation was assessed in two ways; by an artificial circulation system - the Chandler tube system, which measures the rate of formation of platelet aggregates in flowing recalcified citrated plasma, and by a turbidimetric method which measures the ability of platelets in response to adenosine-di-phosphate (ADP). As a measure of platelet adhesion, the diminution in platelet count as a result of passage of citrated whole blood or platelet-rich plasma through a column of glass beads was used (modified Hellem technique). Platelet clumping, as estimated by all methods, was enhanced by both streptokinase and trypsin although the concentrations of each enzyme required to produce this effect varied in the different assay systems. Streptokinase, at a concentration producing maximum lytic activity, accelerated the rate of formation of platelet aggregates in the Chandler tube and produced a marked increase in platelet adhesiveness in the Hellem-type technique. An increase in platelet ADP reactivity was only detected in the turbidimetric system after the plasma antiplasmin had been destroyed. Trypsin, at a concentration of 10 ug/ml sample produced a significant increase in the rate of formation of platelet aggregates in the Chandler tube system and an increase in platelet adhesiveness in the Hellem-type technique, but the concentration of enzyme had to be increased to 100 ug/ml sample before an increase in platelet ADP reactivity was detected. Neither streptokinase nor trypsin influence platelet aggregation by a direct action on the platelet; streptokinase produces no significant enhancement and trypsin produces almost total inhibition of aggregation of washed platelets resuspended in buffer. The effect on platelet aggregation of other proteolytic enzymes -urokinase, ficin, and chymotrypsin was also investigated, but none of these produced any enhancement of platelet reactivity to ADP in the turbidimetric system. These enzymes were however examined in less detail than streptokinase and trypsin. Heparin was also studied and at the concentrations of 0.05 units and 10 units per ml plasma was found to cause significant enhancement of ADP induced! aggregation in the turbidimetric system, but when present at a concentration of 500 units per ml plasma caused significant inhibition of ADP reactivity. The enhancement of platelet aggregation by streptokinase and trypsin was associated with marked fibrinogenolysis; several fibrinogen degradation products were prepared and their effect on platelet aggregation and adhesion examined. The degradation products were prepared by incubation of fibrinogen with either trypsin or urokinase-activated plasminogen followed by fractionation of the protein components of the incubation mixture on a DEAE cellulose column. The ultracentrifugal and electrophoretic properties of the breakdown products were determined and the number of peptide bonds broken during formation of two of the products determined. The effect on platelet aggregation and adhesion was examined of three products of tryptic digestion of fibrinogen; the product of least digestion (molecular weight apprximately 83,000) produced no significant effect on platelet ADP reactivity or platelet adhesiveness, the product of prolonged digestion (molecular weight less than 13,000) inhibited ADP induced platelet aggregation while the product of intermediate digestion (molecular weight 25,000) enhanced the rate of formation of platelet aggregates, ADP reactivity and platelet adhesiveness. A product prepared by digestion with urokinase-activated plasminogen enhanced both the rate of formation of platelet aggregates in the Chandler tube and ADP induced platelet aggregation. The two larger fragments produced by trypsin both exhibited a marked 'antithrombin' effect. The three in vitro methods used to study platelet aggregation and adhesion were compared. No mathematical correlations were found between the results obtained with the different methods; however the effect of trypsin and streptokinase could be detected with each method and the observations made with each method could to some extent be influenced by the concentration of ADP in the system. The experimental data presented demonstrate that under certain circumstances the enzymes streptokinase and trypsin can produce enhancement of platelet aggregation