REGULATION OF HUMAN PLATELET PROTHROMBINASE ACTIVITY

Efforts are focused in two distinct, yet interrelated areas. One goal is to define how the generation of thrombin at the human platelet surface is effected and regulated. Our second goal is to begin to define how thrombin, once formed, interacts with platelet proteins to modulate its activity and the fibrinolytic response. Since thrombin is generated through the proper assembly and function of Prothrombinase at the platelet surface we will test several hypotheses relevant to how functional complex assembly occurs and how the platelet actively regulates these processes. Quantitation of the coordinate binding of factors Va and Xa to platelets followed by assessment of their functional activity under a variety of conditions will allow us to the test the following hypotheses: 1) that Effector Protease Receptor 1 (EPR-1) functions as part of the Prothrombinase receptor(s) at the activated platelet surface; 2) that receptor expression and functional Prothrombinase assembly can be modulated by platelet adherence to extracellular matrix proteins and 3) agonist-induced release and binding of platelet factor V(a) plays a preeminent role in Prothrombinase regulation since platelet factor Va is functionally (and perhaps structurally) different than plasma factor Va with respect to inactivation by proteases such as APC, plasmin, elastase and cathepsin G. The multiple interactions of thrombin with platelet membrane proteins and how they modulate thrombin-induced platelet activation are central to our studies since thrombin activation of platelets has a dramatic effect on the assembly and function of Prothrombinase. Hypotheses suggesting that platelet membrane proteins serve to modulate thrombin activity both positively and negatively will be tested by: 1) identifying and characterizing the platelet's high affinity binding site for thrombin; 2) defining how, and if, thrombin interacts with platelet GPIb or GPIb/IX complexes; and 3) defining the presence and function of thrombomodulin or a thrombomodulin-like molecule at the platelet surface. Finally, we hypothesize that the activated platelet continues to promote a procoagulant response by inhibiting fibrinolysis. To test this hypothesis we will determine if the fibrinolytic response is prolonged through the release of factor Va from the activated platelet as well as the expression of thrombomodulin since both molecules will enhance, albeit through different mechanisms, the thrombin-catalyzed activation of the thrombin activatable fibrinolysis inhibitor (TAFI), a procarboxypeptidase B-like molecule. These combined data indicate that platelets are active participants in both affecting thrombin production and modulating thrombin activity and function. Platelets appear to perform these functions in part through the regulated "receptor"-mediated assembly of proteolytic activities at their membrane surface subsequent to platelet activation. Alternatively, proteins may be released or constitutively expressed which serve to regulate platelet responsiveness and function either positively or negatively. Clearly, several potentially important mechanisms have been discussed here with respect to thrombin production and regulation which require additional study. Since thrombin is an active participant in the hemostatic, thrombotic, and fibrinolytic processes, delineation of these regulatory processes is central to understanding how homeostasis is maintained. Our major accomplishments included demonstrating that: 1) human platelets express a second thrombin receptor/substrate which does not require the anion binding exosite of thrombin for effective interactions; 2) both soluble and cellular forms of thrombomodulin accelerate the thrombin-catalyzed activation of TAFI to effect inhibition of fibrinolysis; 3) the binding of both platelet-derived factor Va and factor VaLeiden to platelets protects them from inactivation by activated protein C; 4) the carbohydrate moieties present on plasma-derived factor V, but not factor Va, regulate its inactivation by activated protein C; and 5) platelet-derived factor V originates from the plasma pool via a megakaryocyte endocytotic mechanism. Our current plans are: 1) to identify and characterize the thrombin high affinity binding site on the platelet membrane surface; 2) to continue to define the functional significance of the platelet membrane protein glycoprotein Ib as it relates to the platelet high affinity binding site; 3) to define the mechanisms by which activated protein C, plasmin and elastase catalyze the activation and/or inactivation of platelet factors V and Va; 4) to develop protocols for the isolation of homogeneous platelet factor V/Va for biochemical characterization and comparison to the plasma-derived protein; and 5) to define the endocytotic pathway by which plasma factor V is transported to the (-granules of megakaryocytes.

Efforts are focused in two distinct, yet interrelated areas. One goal is to define how the generation of thrombin at the human platelet surface is effected and regulated. Our second goal is to begin to define how thrombin, once formed, interacts with platelet proteins to modulate its activity and the fibrinolytic response. Since thrombin is generated through the proper assembly and function of Prothrombinase at the platelet surface we will test several hypotheses relevant to how functional complex assembly occurs and how the platelet actively regulates these processes. Quantitation of the coordinate binding of factors Va and Xa to platelets followed by assessment of their functional activity under a variety of conditions will allow us to the test the following hypotheses: 1) that Effector Protease Receptor 1 (EPR-1) functions as part of the Prothrombinase receptor(s) at the activated platelet surface; 2) that receptor expression and functional Prothrombinase assembly can be modulated by platelet adherence to extracellular matrix proteins and 3) agonist-induced release and binding of platelet factor V(a) plays a preeminent role in Prothrombinase regulation since platelet factor Va is functionally (and perhaps structurally) different than plasma factor Va with respect to inactivation by proteases such as APC, plasmin, elastase and cathepsin G. The multiple interactions of thrombin with platelet membrane proteins and how they modulate thrombin-induced platelet activation are central to our studies since thrombin activation of platelets has a dramatic effect on the assembly and function of Prothrombinase. Hypotheses suggesting that platelet membrane proteins serve to modulate thrombin activity both positively and negatively will be tested by: 1) identifying and characterizing the platelet's high affinity binding site for thrombin; 2) defining how, and if, thrombin interacts with platelet GPIb or GPIb/IX complexes; and 3) defining the presence and function of thrombomodulin or a thrombomodulin-like molecule at the platelet surface. Finally, we hypothesize that the activated platelet continues to promote a procoagulant response by inhibiting fibrinolysis. To test this hypothesis we will determine if the fibrinolytic response is prolonged through the release of factor Va from the activated platelet as well as the expression of thrombomodulin since both molecules will enhance, albeit through different mechanisms, the thrombin-catalyzed activation of the thrombin activatable fibrinolysis inhibitor (TAFI), a procarboxypeptidase B-like molecule. These combined data indicate that platelets are active participants in both affecting thrombin production and modulating thrombin activity and function. Platelets appear to perform these functions in part through the regulated "receptor"-mediated assembly of proteolytic activities at their membrane surface subsequent to platelet activation. Alternatively, proteins may be released or constitutively expressed which serve to regulate platelet responsiveness and function either positively or negatively. Clearly, several potentially important mechanisms have been discussed here with respect to thrombin production and regulation which require additional study. Since thrombin is an active participant in the hemostatic, thrombotic, and fibrinolytic processes, delineation of these regulatory processes is central to understanding how homeostasis is maintained. Our major accomplishments included demonstrating that: 1) human platelets express a second thrombin receptor/substrate which does not require the anion binding exosite of thrombin for effective interactions; 2) both soluble and cellular forms of thrombomodulin accelerate the thrombin-catalyzed activation of TAFI to effect inhibition of fibrinolysis; 3) the binding of both platelet-derived factor Va and factor VaLeiden to platelets protects them from inactivation by activated protein C; 4) the carbohydrate moieties present on plasma-derived factor V, but not factor Va, regulate its inactivation by activated protein C; and 5) platelet-derived factor V originates from the plasma pool via a megakaryocyte endocytotic mechanism. Our current plans are: 1) to identify and characterize the thrombin high affinity binding site on the platelet membrane surface; 2) to continue to define the functional significance of the platelet membrane protein glycoprotein Ib as it relates to the platelet high affinity binding site; 3) to define the mechanisms by which activated protein C, plasmin and elastase catalyze the activation and/or inactivation of platelet factors V and Va; 4) to develop protocols for the isolation of homogeneous platelet factor V/Va for biochemical characterization and comparison to the plasma-derived protein; and 5) to define the endocytotic pathway by which plasma factor V is transported to the (-granules of megakaryocytes.