Homology model of human prothrombinase based on the crystal structure of Pseutarin C

Abstract

Thrombin is generated from prothrombin through cleavage at two sites by the prothrombinase complex. Prothrombinase is composed of a protease, factor (f) Xa, and a cofactor, fVa, which interact on negatively charged phospholipid surfaces and cleave prothrombin into thrombin 300 000 times faster than fXa alone. The balance between bleeding and thrombosis depends on the amount of thrombin produced, and this in turn depends on the function of the prothrombinase complex. How fXa and fVa interact and how improved prothrombin processing is conferred are of critical importance for understanding healthy and pathological blood clotting. Until recently, little structural information was available, and molecular models were built on partial structures with assembly guided by biochemical data. Last year our group published a crystal structure of a prothrombinase complex from the venom of the Australian Eastern Brown snake (known as Pseutarin C). Here we use the crystal structure of Pseutarin C as a starting point for homology modelling and assembly of the full human prothrombinase complex. The interface is complementary in shape and charge, and is consistent with much of the published biochemical data. The model of human prothrombinase presented here provides a powerful resource for contextualizing previous data and for designing future experiments.

Blood coagulation factor Va's key interactive residues and regions for prothrombinase assembly and prothrombin binding

“…Interestingly, the crystal structure of pseutarin C reveals that these residues are located at a relatively far distance from FXa (>20 Å). In pseutarin C, only Met318, homologous to human Arg317, was found to interact with FXa, and similar observations were made for recent homology models of human prothrombinase (Figure ; Table S1). It therefore seems unlikely that the A2 domain Arg306 region in FVa is directly contributing to FXa binding, but rather seems to be involved in the interaction with prothrombin (see also Section 4).…”

“…As pseutarin C shares ~40% to 60% sequence identity with human FVa‐FXa, this crystal structure provides a reliable blueprint for molecular modeling of human prothrombinase. Consequently, the latter was generated by Huntington and coworkers . More recently, a full human ternary complex was reported, thereby providing important new insights in the structural organization of the prothrombinase complex and prothrombin binding.…”

“…Structural analysis confirms that these residues are all located on the A2 domain surface in close proximity to FXa (<4 Å) (Figure ). Furthermore, these residues and their homologs in pseutarin C have been implicated as FXa contact residues …”

“…Furthermore, alanine substitution of residues Arg1551, Glu1650, Trp1665, and His1683 in FVa resulted in a minor reduction in FXa affinity . The aforementioned residues have also been identified as FXa contact residues in several prothrombinase structures . Within the pseutarin C FV sequence Thr789‐Gln922 (equivalent to human FV Ser1546‐His1683), seven residues were found to be directly involved in FXa binding, while 16 other amino acids were considered as contact residues .…”

TFPIα interacts with FVa and FXa to inhibit prothrombinase during the initiation of coagulation

“…For example, a peptide mimicking the FV B-domain basic region, which is homologous to the TFPIα basic region, competes with FXa for binding to FV810. 21 Because FXa binds the FVa heavy chain, 36,37 it has been proposed that the basic peptide binds the FVa heavy chain and inhibits prothrombinase assembly. 21 This mechanism has since been extrapolated to full-length TFPIα, which is being described as an inhibitor of prothrombinase assembly.…”