Identification and characterization of a factor Va-binding site on human prothrombin fragment 2

Abstract

The fragment 2 domain (F2) of prothrombin and its interaction with factor (F) Va is known to contribute significantly to prothrombinase-catalyzed activation of prothrombin. The extent to which the F2-FVa interaction affects the overall thrombin generation, however, is uncertain. To study this interaction, nuclear magnetic resonance spectroscopy of recombinant F2 was used to identify seven residues within F2 that are significantly responsive to FVa binding. The functional role of this region in interacting with FVa during prothrombin activation was verified by the FVa-dependent inhibition of thrombin generation using peptides that mimic the same region of F2. Because six of the seven residues were within a 9-residue span, these were mutated to generate a prothrombin derivative (PT6). These mutations led to a decreased affinity for FVa as determined by surface plasmon resonance. When thrombin generation by an array of FXa containing prothrombinase components was monitored, a 54% decrease in thrombin generation was observed with PT6 compared with the wild-type, only when FVa was present. The functional significance of the specific low-affinity binding between F2 and FVa is discussed within the context of a dynamic model of molecular interactions between prothrombin and FVa engaging multiple contact sites.

Disruption of the kringle 1 domain of prothrombin leads to late onset mortality in zebrafish

“…Quantitative real-time PCR. Total RNA was extracted from 7 days post fertilization larvae (dpf) using the RNeasy Mini Plus kit (Qiagen) and transcribed using oligo(dT) [12][13][14][15][16][17][18] primer and Superscript III (Invitrogen). Three pools of three whole larvae were used per genotype.…”

“…Excitation and emission spectra were 280 nm and 540 nm, respectively, with an emission cutoff filter set at 530 nm. The quantum yield of the thrombin-DAPA complex was determined by plotting the total signal change observed with respect to known concentrations of the thrombin-DAPA complex 12 .…”

High Mutational Heterogeneity, and New Mutations in the Human Coagulation Factor V Gene. Future Perspectives for Factor V Deficiency Using Recombinant and Advanced Therapies

“…This wide range of mutational changes observed in the F5 gene is related to a large number of highly variable effects at a functional and molecular level. Some of the consequences of the different mutations include alterations in the stability of FVa [ 61 ]; alterations in the splicing [ 62 , 63 ] and in the biogenesis of FVa [ 64 , 65 , 66 , 67 ], mainly due to secretion disturbances; and mutations affecting the binding sites of the protein to certain activation or inactivation factors [ 68 ]. The mutations described in this study are related to the appearance of the stop codon, which results in a non-functional truncated protein.…”

A Novel Heterozygous Variant in F2 Gene in a Chinese Patient With Coronary Thrombosis and Acute Myocardial Infarction Leads to Antithrombin Resistance

“…Our study may be the second report that a mutation of F2 may also lead to coronary thrombosis and acute myocardial infarction. Coagulation factor II is proteolytically cleaved to form thrombin in the first step of the coagulation cascade which ultimately results in the stemming of blood loss (Friedmann et al, 2019). F2 also plays a role in maintaining vascular integrity during development and postnatal life (Sun et al, 2002).…”