Bovine Platelets in Large Quantities Properties and Activities Concerned With Hemostasis

Abstract

Platelets of a high degree of purity were prepared in 10-20 g batches from oxalated blood of cows Bos taurus, collected during post mortem exsanguination. Platelet-rich plasma was collected in a continuous flow process using the blood bowl of the Sharpies centrifuge, and a platelet concentrate was obtained from this as a thick paste by a second centrifugation in a clarifier bowl. Platelets of any desired degree of purity could be obtained from the concentrate by repeated washing with saline, with conventional centrifugation to further remove erythrocytes and leucocytes. Three washes, together with a final packing, were estimated to dilute out free plasma proteins to 10-8; leucocytes were reduced to I/million platelets. Greatest loss of platelets occurred during separation of the platelet-rich plasma; final yield about 30%. Platelet properties of " stickiness", agglutination, and susceptibility to lysis were much altered. Many, but not all, of the platelets of a suspension could be disrupted by freezing (efficient reduction could be achieved with a sonic generator). The platelets were sufficiently pure to be used as a source of platelet reagent for evaluation of the platelet-plasma "thromboplastin-equivalent" mechanism of initiation of blood coagulation (thrombokinase mechanism). Several other factors, including the vasoconstrictor serotonin, a clot retraction agent, a clottable substance and an antifibrino-lysin were present. The particulate platelet debris, remaining after thawing, was the most useful reagent derived from platelets; it could be washed by resuspension and again centrifuged out of suspension; and could be likewise removed from a reaction mixture during or after the reaction. This was useful in analysis of the coagulation defect of hemophilia, and was effective for the quantitative conversion of prothrombin to thrombin in a purified system, but only after a lag phase of 3-5 minutes. The platelet cofactor for reaction with a plasma cofactor to generate a " thromboplastin-equivalent" (thrombokinase) resided within the particulate portion of this reagent. By contrast to tissue thrombo-plastin, there was a remarkable physiologic margin of safety against the platelet thrombokinase mechanism when platelet extract was injected in vivo.