Platelets Contribute to Protein S Cleavage In Vivo.

Protein S, a vitamin K-dependent plasma protein, is one of the molecules involved in down-regulation of the coagulation process. Protein S serves as a cofactor of activated protein C (APC) in the proteolytic inactivation of activated factor V and VIII. Protein S is also able to exert its anticoagulant activity independent of APC, e.g. by supporting the anticoagulant activity of tissue factor pathway inhibitor. In plasma, protein S circulates in two forms: a single chain molecule with full anticoagulant activity and a two-chain form with reduced anticoagulant activity. This two-chain variant is the result of cleavage in the protease sensitive loop, the so-called thrombin sensitive region (TSR). The enzyme responsible for the presence of cleaved protein S in the circulation is not known yet. In vitro, thrombin and factor Xa, both enzymes of the coagulation cascade, are able to cleave the TSR. In a recent study (Brinkman et al, J Thromb Haemost 2005; 3: 2712-2720), it was shown that these enzymes do not cleave protein S under physiological conditions. Instead, a platelet protease was found to cleave protein S in plasma during tissue factor induced clotting in vitro. We hypothesize that this platelet protease may be responsible for the presence of cleaved protein S in the circulation.

To correlate cleaved protein S levels in the circulation with the activity of platelet-associated protein S cleaving protease and to evaluate the impact of protein S cleavage on its anticoagulant activity in terms of total protein S activity.

Protein S cleavage was evaluated by immunological methods employing a monoclonal antibody specific for uncleaved, single chain protein S. The total anticoagulant activity of protein S (APC-independent and APC-dependent) was assessed employing thrombography.

We observed in the in vitro thrombin generation test massive generation of thrombin in protein S deficient plasma that was dose-dependently inhibited by intact, single chain protein S. TSR-cleaved protein S had totally lost its anticoagulant activity. In plasma from healthy volunteers, cleaved protein S expressed as % of the total amount of protein S ranged between 8 and 51% with an average normal value of 26 ± 9% (mean ± SD, n=46). Levels of intact protein S correlated well with total protein S activity in the individual plasma samples, while this correlation was not observed for levels of TSR-cleaved protein S. These data suggest that not only in vitro but also in vivo, cleavage of protein S has an impact on its anticoagulant activity. A significant reduction of levels of cleaved protein S was observed in chemotherapy induced thrombocytopenia in hematological patients (see Figure). At a platelet count < 50, the average protein S cleavage was dropped to 16 ± 7% (mean ± SD, n=15). Upon platelet transfusion, cleavage of protein S dramatically increased to 36 ± 11% (mean ± SD, n=11). Levels of total protein S were similar in healthy volunteers and thrombocytopenic patients before and after platelet transfusion. It thus appears that platelets contribute to protein S cleavage in vivo. However, in healthy individuals showing a wide range of protein S cleavage (see Figure), no correlation was observed between platelet count (200-400 .10⁹/l) and protein S cleavage. This suggests additional sources of protease that cleave protein S in vivo. We therefore examined lysates of a variety of cell types for protein S proteolytic activity. Protein S proteolytic activity was absent in lysates of erytrocytes, vascular smooth muscle cells and fibroblasts. Lysates of HUVEC did show protein S proteolytic activity similar to platelets.

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Our results strongly suggest that platelets contribute to the proteolytic modification of protein S in vivo resulting in an abolished anticoagulant activity. An additional source of protein S proteolytic activity may be the endothelium. Cleavage of protein S provoked by platelet infusion may contribute to the beneficial effect of platelet transfusion in the treatment of bleeding episodes.

No relevant conflicts of interest to declare.