Crystallization of Glutaraldehyde Cross-Linked Prothrombinase Complex in its Active Form

Abstract

Activation of prothrombin to thrombin is catalyzed by “prothrombinase” complex, consisting of factor Xa and its cofactor factor Va bound to phosphatidylserine (PS)-containing membrane. While partial crystal structures of Xa and Va exist, attempts to crystallize XaVa complex failed. Recent studies shows that both Xa and Va2, an active isoforms of Va binding tightly to PS-membrane, binds soluble dicaproyl-phosphatidylserine (C6PS). Also, C6PS-bound form of Va2 binds with high affinity (Kd ∼ 1 nM) to Xa forming a fully active prothrombinase complex in solution. Preliminary studies show this soluble complex, when cross-linked with glutaraldehyde, remains intact after removing C6PS using size-exclusion chromatography. Depending on conditions, up to 92% of initial activity is restored after adding back C6PS. To determine optimal formation conditions of an active cross-linked XaVa complex for use in crystallization trials, we used an algorithm varying Xa/Va and glutaraldehyde concentration, incubation time, and temperature. XaVa activity was tested after four critical stages (complex formation, cross-linking with glutaraldehyde, removing C6PS, reactivation with C6PS) by a chromogenic assay, and the yield of cross-linked complex was determined by using quantitative Shodex size-exclusion chromatography. While preliminary experiments were performed using plasma-derived human Va2, greater quantities of protein are needed than can be produced by this method. Since crystallization is more successful with cloned recombinant proteins, our lab works with multiple labs to establish procedures for expressing large quantities of recombinant human Va2 (rHVa2) in Baby Hamster Kidney cells (BHK). With higher yields of Va2, we are proceeding with trials to optimize conditions for cross-linked active prothrombinase complex. If these are successful, we will continue with crystallization trials, enabling us for the first time to determine the structure of XaVa complex in its active form. Supported by grant HL072827 and a training supplement thereto.