Recent Research Developments in the Direct Inhibition of Coagulation Proteinases – Inhibitors of the Initiation Phase

Abstract

Physiologic clotting is a defensive action. The new cell-based model of hemostasis proposes three steps -initiation, amplification and propagation -occurring on specific cell surfaces to generate a thrombus in a tightly regulated manner. The initiation phase relies on key players including tissue factor (TF), factor VIIa (fVIIa), platelets, Ca 2+ , phospholipids, and factor X/Xa (fX/fXa). Exposure of TF on sub-endothelial and other blood cells triggers a coagulation response, which may have to be inhibited to prevent a deleterious thrombotic effect. Inhibiting TF-initiated coagulation, akin to 'nipping coagulation in the bud', is predicted to have major advantages, including a more efficient separation of the antithrombotic and hemorrhagic responses. The availability of crystal structures of TF, fVIIa and TFfVIIa complex makes structure-based drug design feasible. Although no initiation phase small molecule inhibitor has reached the clinic as yet, several molecules have displayed promise. We discuss recent results on the discovery of inhibitors of the initiation phase with special emphasis on peptides, peptidomimetics and organic small molecules.Key Words: Anticoagulants, coagulation, factor VIIa, tissue factor, thrombin, factor Xa, Enzyme Inhibition, small molecule inhibitors. CELL-BASED HEMOSTASISPhysiologic clotting is a defensive action preventing excessive loss of blood and infiltration of microbes. The physiologic mechanism that produces this defensive reaction is an enzymatic cascade ( Fig. 1), proposed nearly 50 years ago [1,2]. In vivo, the enzymatic reactions occur on specific cell surfaces. The cell-dependent coagulation cascade is proposed to proceed sequentially in three stepsinitiation, amplification and propagation -on specific cell surfaces to generate a thrombus in a tightly regulated manner [3][4][5]. InitiationTissue factor (TF) is an integral membrane protein present on several extravascular cells [6] and is the most important initiator of coagulation. Under normal conditions, it is sequestered from circulating proteinases, especially factor VII (fVII), thus preventing inappropriate initiation of clotting. Vascular injury exposes TF to blood, resulting in rapid complex formation with free activated fVII (fVIIa) on TF-bearing cells (Fig. 1). It is believed that some fVIIa is always present in blood (~1-2%), which can rapidly interact with TF to initiate clotting. The formation of TF/fVIIa complex activates the circulating factor X (fX) to fXa, the formation of which can induce further production of fVIIa and fXa [7][8][9]. The TF/fVIIa complex can also activate factor IX (fIX) to fIXa [10][11][12], which in turn can result in more fXa. The earliest fXa formed remains localized to the site of TF-bearing cells, yet is free enough to form the prothrombinase complex with factor Va (fVa). The small amount of the prothrombinase complex so formed is responsible for the initial production of thrombin from prothrombin [12]. The in vivo source of the initial fVa needed for the assembly of p...

Interaction of Antithrombin with Sulfated, Low Molecular Weight Lignins

“…The mechanistic basis for SDSO3 activation of antithrombin inhibition of free fVIIa is not clear at the present time, but it probably arises from the modulation of the reactive center loop conformation due to binding in the HBS. This presents an interesting opportunity for regulating the cellular initiation phase of hemostasis (47,48).…”

Identification of the site of binding of sulfated, low molecular weight lignins on thrombin

“…The combination of a hydrophobic, aromatic scaffold and selected number of charged groups in sulfated LMWLs induces novel physicochemical and protein recognition properties [7-9]. In this respect, sulfated LMWLs are proving to be unlike any other class of anticoagulants being investigated to-date, including the heparins, the coumarins, the hirudins, the peptidomimetics and the small molecule direct inhibitors [2,10]. …”

Dynamic affinity chromatography in the separation of sulfated lignins binding to thrombin

“…Clotting assays are typically used to assess coagulation state of plasma. The coagulation cascade is traditionally divided into intrinsic and extrinsic pathways, which are comprised of distinct coagulation factors, although it is well established that both pathways are highly intertwined with multiple feedback and feed-forward loops [6]. Traditionally, PT measures the state of extrinsic pathway, which is primarily comprised of factor VII and tissue factor; while APTT relates more with the intrinsic pathway, which includes factors IX, XI and XII.…”

“…An overwhelming number of orthosteric inhibitors of thrombin and factor Xa have been designed [6,14,15] and our discovery that sulfated LMWLs utilized a radically different mechanism [3] suggested a strong possibility of uncovering potent oligomeric allosteric anticoagulants.…”