UVM Theses and Dissertations
Format:
Print
Author:
Whelihan, Matthew F.
Dept./Program:
Biochemistry
Year:
2013
Degree:
PhD
Abstract:
Thrombin generation occurs via an integrated enzymatic cascade that involves plasma, tissue and cellular components. Prothrombin activation by prothrombinase (Ilase; factor (F)Xa·FVa) involves cleavage at Arg 271 and Arg 320 to produce the [alpha]-thrombin ([alpha]lIa) product. Depending on the order of cleavage, activation occurs via two possible intennediates: meizothrombin (mIla) or prethrombin-2. mIla arises from initial cleavage at Arg 320 yielding a membrane-binding enzyme; initial cleavage at Arg27l yields an inactive non-covalent complex of fragment 1.2 and prethrombin-2. Cleavage at the alternate Arg residue yields ulla. On synthetic phospholipid vesicles, prothrombin activation by Ilase proceeds exclusively through the mIla pathway, with the probable mechanism involving mIla dissociating from the I1ase complex and then rebinding to Ilase to yield [alpha]IIa.
On washed platelets, prothrombin activation proceeds through the prethrombin-2 pathway, with no detectable mIla being released from the platelet surface. However, mIla has been observed in clotting blood, suggesting that prothrombin activation in blood involves both cleavage pathways. The relative prevalence ofthese two pathways in blood has potentially interesting regulatory consequences for hemostasis.
Platelets have been traditionally viewed as the primary cellular component playing an active role in thrombin generation. Upon activation by thrombin and other activators, platelets support binding of the coagulation complexes (intrinsic factor Xase and IIase) on their membranes which serves to localize thrombin generation near the site of vascular injury. Furthennore, the activated platelet surface has been shoWn to optimize procoagulant activity via modulation of the pathway of thrombin generation. In purified systems, physiologic levels (l50-400xl0⁶/mL) of activated platelets are capable of generating 60-150 nM/min thrombin. Thus it was assumed that platelets were the primary cell surface upon which thrombin generation occurs.
In general, red blood cells (RBCs) have not been thought to play a significant mechanistic role in normal hemostasis and have been considered "innocent bystanders" in the clotting process. The commonly accepted model for the hemostatic process following vessel injury involves initiation triggered by simultaneous exposure of extravascular proteins, specifically tissue factor and extracellular matrix proteins that promote platelet adhesion and activation. For the past 100 years, clinical data has shown that a wide variety of bleeding disorders such as thrombocytopenia, iron deficiency anemias, anemic uremias and platelet adhesion defects can be successfully treated simply by elevation of RBC counts. These platelet-independent effects point to RBCs as playing an important role in hemostasis. In addition to the identification of the source of mIla production in whole blood, this research project supports a biochemical role for RBCs in coagulation. Thus in TF-activated blood, RBCs and platelets both appear to play significant roles in thrombin generation.
On washed platelets, prothrombin activation proceeds through the prethrombin-2 pathway, with no detectable mIla being released from the platelet surface. However, mIla has been observed in clotting blood, suggesting that prothrombin activation in blood involves both cleavage pathways. The relative prevalence ofthese two pathways in blood has potentially interesting regulatory consequences for hemostasis.
Platelets have been traditionally viewed as the primary cellular component playing an active role in thrombin generation. Upon activation by thrombin and other activators, platelets support binding of the coagulation complexes (intrinsic factor Xase and IIase) on their membranes which serves to localize thrombin generation near the site of vascular injury. Furthennore, the activated platelet surface has been shoWn to optimize procoagulant activity via modulation of the pathway of thrombin generation. In purified systems, physiologic levels (l50-400xl0⁶/mL) of activated platelets are capable of generating 60-150 nM/min thrombin. Thus it was assumed that platelets were the primary cell surface upon which thrombin generation occurs.
In general, red blood cells (RBCs) have not been thought to play a significant mechanistic role in normal hemostasis and have been considered "innocent bystanders" in the clotting process. The commonly accepted model for the hemostatic process following vessel injury involves initiation triggered by simultaneous exposure of extravascular proteins, specifically tissue factor and extracellular matrix proteins that promote platelet adhesion and activation. For the past 100 years, clinical data has shown that a wide variety of bleeding disorders such as thrombocytopenia, iron deficiency anemias, anemic uremias and platelet adhesion defects can be successfully treated simply by elevation of RBC counts. These platelet-independent effects point to RBCs as playing an important role in hemostasis. In addition to the identification of the source of mIla production in whole blood, this research project supports a biochemical role for RBCs in coagulation. Thus in TF-activated blood, RBCs and platelets both appear to play significant roles in thrombin generation.