The Interaction of Prothrombin and Its Activation Products with Platelets

• Published in: Blood Vol. 108; no. 11; p. 1514
• Main Authors: Samis, John A., Manuel, Reginald P., Nesheim, Michael E.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 16.11.2006
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V108.11.1514.1514

The interaction of human 125I-prothrombin and its activation products with unactivated and thrombin-stimulated human platelets was studied. 125I-prothrombin binding to unactivated platelets was found to be a reversible and calcium-dependent process (n=48,000 sites/platelet, Kd=3.0μM). Thrombin stimulation of platelets resulted in increased prothrombin binding both in the presence and absence of calcium (n=84,000 sites/platelet, Kd=6.0μM and n=53,000 sites/platelet, Kd=8.0μM, respectively). Thrombin stimulation of platelets also increased the calcium-independent binding of prethrombin-1 compared with unstimulated cells. Using thrombin-stimulated platelets as a membrane surface for prothrombinase, 125I-prothrombin was converted exclusively to 125I-Fragment 1.2 and 125I-thrombin when the reactions were carried out in the presence of the reversible thrombin inhibitor, 5-dimethylaminonapthalene-1-sulfonylarginine-N-(3-ethyl-1,5-pentanediyl)amide (DAPA). 125I-thrombin was the major prothrombin activation product that remained bound to the thrombin-stimulated platelets (50,000 molecules/platelet). Platelet-bound 125I-thrombin either added directly or generated from 125I-prothrombin by the action of prothrombinase was found to be inacessible to human antithrombin (AT)/heparin-induced inactivation. Utilization of phosphatidylcholine/phosphatidylserine (PCPS) vesicles as a membrane surface for prothrombinase in the presence of DAPA demonstrated that 125I-prothrombin activation proceeded initially through a meizothrombin intermediate that was replaced at later times by Fragment 1.2 and the A and B chains of α-thrombin. 125I-Fragment 1.2 was the main prothrombin activation product that remained bound to the PCPS vesicles after Sephadex G-150 gel filtration chromatography. The above results indicate that human prothrombin binds specifically to unstimulated human platelets via its Fragment 1 domain only in the presence of calcium and thrombin-stimulation results in the exposure of additional calcium-dependent and -independent prothrombin binding sites. In addition, thrombin was the major prothrombin activation product that remains associated with the thrombin-stimulated human platelets and this platelet-bound thrombin is protected from AT/heparin- induced inactivation. During prothrombin activation on platelets, large amounts of thrombin remain bound and Fragment 1.2 is released from the platelet surface. In contrast, Fragment 1.2 was the major prothrombin activation product that binds PCPS vesicles. Therefore, although both thrombin-stimulated human platelets and PCPS vesicles may each serve as an effective membrane component of prothrombinase, they appear to differ both in their prothrombin activation pathway as well as their ability to bind the products of prothrombin activation.