The Heeb Laboratory

Research

Our lab investigates anticoagulant plasma proteins that regulate blood coagulation and prevent thrombosis. These include protein S, protein Z, and protein Z-dependent protease inhibitor, which downregulate factor (F) Xa and other targets. Heterozygous deficiency of protein S or protein Z is associated with increased risk of stroke and venous thrombosis. Thus, knowledge of molecular mechanisms underlying the anticoagulant activities of these proteins may lead to new antithrombotic therapies.

Protein S is known as a cofactor for the anticoagulant, activated protein C (APC). However, we reported that protein S also has APC-independent, direct anticoagulant activity (PS-direct) via inhibition of FXa and FVa. We validated PS-direct in a baboon thrombosis model, where protein S effectively curbed platelet and fibrin deposition, even when APC was blocked. Therefore, protein S shows promise as an antithrombotic drug.

We found that protein S isolated by commonly-used conventional methods had much weaker PS-direct than immunoaffinity-purified protein S in the baboon thrombosis model. In clotting assays, protein S-depleted plasma exhibited a shorter lag time before thrombin generation and greater overall thrombin generation than normal protein S-containing plasma. Immunoaffinity-purified protein S reconstituted protein S-depleted plasma, diminished thrombin generation, and increased the lag time, but conventionally-purified protein S did not. We investigated the source of variability in PS-direct of protein S in order to reveal a protein S feature that is crucial for PS-direct.

We found that immunoaffinity-purified protein S contained 1 Zn²⁺ atom/molecule, while conventionally-purified protein S contained 0-0.3 Zn²⁺ atom/molecule. Zn²⁺ content correlated with PS-direct and we demonstrated reversible loss/regain of PS-direct with loss/gain of Zn²⁺. A Zn²⁺ binding site was postulated at the interface of two laminin G-type domains near the C-terminus of protein S. Zn²⁺ may be essential for interdomain interactions required for PS-direct, and site-directed mutagenesis underway may confirm this location. Antibodies directed against a site at the interdomain interface efficiently recognized only protein S that contained Zn²⁺, suggesting differences in conformation between Zn²⁺-deficient and Zn²⁺-containing protein S.

We are defining which molecular interactions of protein S are affected by Zn²⁺ occupancy. FXa binds to Zn²⁺-containing protein S with 12X greater affinity than it does to Zn²⁺-deficient protein S. Yet, tissue factor pathway inhibitor (TFPI) binds almost equally well to either type of protein S, suggesting a TFPI binding site on protein S outside of the Zn²⁺-containing region. APC cofactor activity of protein S is also unaffected by Zn²⁺-content. The N-terminal region of protein S is largely responsible for APC cofactor activity, further suggesting a location for Zn²⁺ near the C-terminus. Detailed investigation of the activities and binding behavior of protein S toward these molecules and FVa as a function of Zn²⁺ content is underway. Other studies address a possible biological role for the interaction between protein S and TFPI. Overall, Zn²⁺ may partially regulate PS-direct.

On a related topic, ten neonates with life-threatening thrombosis have been reported who had compound heterozygous protein S deficiency. Remarkably, we identified a compound heterozygous young adult who did not experience recurrent thrombosis until age 10. His protein levels were 10% of normal. All protein S was in the form of protein S-C4b-binding protein complexes, a form with poor APC cofactor activity. Thus, PS-direct may have been partially protective in this individual.

In collaboration with Salk investigators, a protein S knock-out mouse has been developed. Novel assays were developed for mouse protein S antigen and the two major types of protein S anticoagulant activity in mice, assays that will also be useful in future mouse model studies. The knock-out project to date dramatically illustrates the importance of protein S in preventing thrombosis through both PS-direct and APC cofactor activity.