Relationship between platelet secretion and prothrombin cleavage in native whole blood

Abstract

To determine the relationship between platelet secretion and prothrombin conversion in whole blood, the release of platelet factor 4 and the generation of a X(a)-specific cleavage product of prothrombin, fragment 1+2, were measured during the coagulation of whole blood. There was a parallel increase in the concentration of the two proteins. Over the first 5 min of incubation, platelet factor 4 concentration increased 6 ng/ml per min, and after 6-7 min,the rate of release increased to 750 ng/ml per min. Over the initial 5-7 min of incubation, fragment 1+2 concentration increased 1.5 pmol/ml per min with a subsequent increase of 45 pmol/ml per min. Incubation with 10 μM prostaglandin E1 or 15 μM prostaglandin I2 inhibited secretion of platelet factor 4 and delayed the onset of the rapid phase of fragment 1+2 generation by 8 min, while stimulation of platelet secretion with 1 μg/ml collagen suspension enhanced production of fragment 1+2. The addition of either 10 μM epinephrine or 100 ng/ml collagen suspension to whole blood did not affect either platelet factor 4 release or fragment 1+2 generation, although the combination of 3 μM epinephrine and 100 ng/ml collagen suspension enhanced platelet release and prothrombin cleavage. The relationship between platelet factor 4 release and prothrombin cleavage was also studied in Factor VIII-deficient blood. When 0.001 U/ml factor VIII activity was present, <80 ng/ml platelet factor 4 were released, and no fragment 1+2 was generated after 30 min of incubation. The addition of 0.008-0.08 U/ml Factor VIII activity progressively increased platelet factor 4 release and prothrombin cleavage. Platelet factor 4 release was normal at 0.08 U/ml Factor VIII activity, whereas prothrombin cleavage was still delayed. Very little thrombin, the amount generated by the cleavage of 3-5 nM fragment 1+2, was needed to induce release of platelet factor 4.