Cyclooxygenases (COX, PGH synthases) are key enzymes in the biosynthesis of prostaglandins and thromboxane. Two isoenzymes exist with a 61% of sequence identity, which are products of different genes. COX-1, considered as a constitutive version of the enzyme, is mainly responsible for housekeeping functions [1]. COX-2, the second isoform, is inducible in most tissues and associated with cellular stress (e.g., shear stress in vascular tissue), inflammatory processes, and cell proliferation. Platelets largely express the COX-1 isoform.

Structure, expression, and catalytic activity of platelet COX-1

Both COX isoforms consist of two identical heme-containing subunits inserted in the endoplasmic membrane. The substrate arachidonic acid is bound in a channel extending into the interior of the protein where it is converted by two sequential reactions into prostaglandin G₂ (PGG₂) and prostaglandin H₂ (PGH₂). The first reaction (at the COX site) inserts two oxygen molecules into the substrate fatty acid and catalyzes a cyclization of the carbon backbone. The product, PGG₂, is transformed at a different site (peroxidase site) to PGH₂, which involves the reduction of the 15-hydroperoxide group of PGG₂. In platelets, PGH₂ is further converted by thromboxane synthase into thromboxane A₂ (TXA₂).

The preferred substrate of COX-1 and COX-2 is arachidonic acid, which is released by phospholipase A₂ (PLA₂) from the glycerophospholipids of cell membranes. Ceramide kinase and ceramide-1-phosphate contribute by bioactivation of cytosolic PLA₂. Alternative sources of arachidonic acid are arachidonylethanolamide (anandamide) and 2-arachidonylglycerol [2].

COX activity requires an activating hydroperoxide to generate a tyrosyl radical at Tyr385, which is essential to initiate catalytic activity. Thus, platelet TXA₂ formation also depends on the concentration of COX-activating lipid peroxides. The local activity of peroxides is particularly high in platelets, allowing for an extensive burst of PGH₂ and TXA₂ synthesis when platelets are activated [3]. A possible consequence in the intact vasculature may be that vascular PG formation (e.g., PGI₂) is more completely suppressed by NSAIDs than platelet TXA₂ synthesis.

Functional role of platelet COX-1

COX-1-deficient mice have reduced arachidonic acid-induced platelet aggregation [4], confirming a wealth of experimental data demonstrating that COX-1 is critical for platelet activation. In addition, nonplatelet COX activity in vascular tissues also regulates vascular function and thrombosis. Hence, cardiovascular effects of NSAIDs depend on the inhibition of platelet TXA₂ via platelet COX-1 and on inhibition of vascular PG formation by extraplatelet COX-1 and COX-2. Their products (e.g., PGI₂ and TXA₂) have opposing biological effects on vasculature (vascular tone, thrombogenicity, growth) and platelets (aggregation, secretion).

Genetic polymorphisms of COX-1 and COX-2 expression in platelets

Sequence analysis of COX-1 has identified genetic variations that alter COX-1 activity (K185T, G230S, L237M) and change COX-1 sensitivity to indomethacin (P17L, G230S) [5]. Another COX-1 variant (G-1006A) has been associated with an elevated risk of ischemic stroke [6]. However, data on the importance of COX polymorphisms for platelet function and platelet sensitivity to aspirin are inconsistent [7].

Several years ago, our laboratory has demonstrated that platelets may also contain the inducible COX isoform COX-2 [8]. Subsequent work from others showed that COX-2 is required for megakaryocyte differentiation [9]. Thus, it is conceivable that COX-2 message and protein are carried over into the mature platelets. Some authors suggested that platelet COX-2 may bypass COX-1 and result in an impairment of the antiplatelet action of aspirin due to the lower sensitivity of COX-2 toward aspirin [10], while others did not detect COX-2-dependent TXA₂ formation by human platelets at all [11]. Further work identified COX-2 mRNA in platelets as a COX-2 variant (COX-2a) with a loss of about 100 bp in exon 5 [12]. The deduced protein was metabolically inactive [13].

Platelet COX-1 as a target for antithrombotic therapy

The usefulness of aspirin for first-line antiplatelet therapy to prevent atherothrombotic complications in vascular disease is well established. This is covered by Chapter 13.

Unlike aspirin, the naNSAID-induced inhibition of platelet COX-1 depends on the half-life in plasma, which is relatively short (few hours) for most compounds. NaNSAIDs also act competitively, allowing the local concentration of arachidonic acid to displace the compounds from their binding sites within the COX-1 substrate channel. Due to a nonlinear relationship between platelet TXA₂ generation and function [14], decreasing naNSAID plasma levels result in rapid and full recovery of platelet function (Figure 2.1A). Thus, naNSAIDs are not appropriate for circadian platelet inhibition.

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