Protein S and factor V in regulation of coagulation on platelet microparticles by activated protein C

Abstract

Introduction

Platelets are the main source of microparticles in plasma and the concentration of microparticles is increased in many diseases. As microparticles expose negatively charged phospholipids, they can bind and assemble the procoagulant enzyme-cofactor complexes. Our aim was to elucidate possible regulation of these complexes on microparticles by the anticoagulant protein C system.

Materials and methods

Platelets were activated with thrombin ± collagen or the calcium ionophore A23187 ± thrombin to generate microparticles. The microparticles were analyzed using flow cytometry and functional coagulation assays to characterize parameters with importance for the activated protein C system.

Results

Activation with A23187 + thrombin was most efficient, fully converting the platelets to microparticle-like vesicles, characterized by high lactadherin and protein S binding capacity. Suppression of thrombin generation by activated protein C in plasma spiked with these microparticles was dependent on the presence of plasma protein S. Experiments with purified components showed that activated protein C inhibited both factor Va and factor VIIIa on the microparticle surface. Inhibition of factor Va was stimulated by, but not fully dependent on, the presence of protein S. In the factor VIIIa-degradation, activated protein C was dependent on the addition of protein S, and exogenous factor V further increased the efficiency.

Conclusions

Protein S is crucial for activated protein C-mediated inhibition of thrombin generation on platelet-derived microparticles in plasma. Moreover, protein S and factor V are synergistic cofactors in the inhibition of factor VIIIa. The results demonstrate that the activated protein C system has the capacity to counterbalance the procoagulant ability of microparticles.

Introduction

Microparticles (MPs) are small membrane-containing vesicles released by numerous cell types upon activation, apoptosis or stress [1], [2], [3]. Their features are dependent on the cell origin; hence they can be identified by surface expression of cell specific markers. MPs from platelets have a high surface content of the negatively charged phospholipid phosphatidylserine, and support the activations of factor X (FX) and prothrombin [4], [5], [6], [7], [8]. FX is activated by the intrinsic Xase complex where activated factor IX (FIXa), together with its cofactor activated factor VIII (FVIIIa), is bound to negatively charged phospholipids, whereas prothrombin is activated by the prothrombinase (PTase) complex (activated FX (FXa), its cofactor activated factor V (FVa) and the phospholipids). The platelets contain about 20% of the total amount of FV in blood [9]. This platelet-derived FV is released upon activation and bind to the negatively charged activated platelets, thereby contributing to the formation of PTase complexes. The fully assembled complexes are 10⁵-10⁶ more efficient than the respective enzymes alone [10], [11].

The Xase- and PTase complexes are regulated by activated protein C (APC) [12], which inactivates FVIIIa and FVa [13], [14]. Protein S serves as an APC-cofactor in these reactions [15], [16]. In human plasma, approximately 35% of protein S is free, the remaining being bound to C4b-binding protein (C4BP) [17]; mainly the free form serving as APC cofactor [18]. In the inhibition of FVIIIa, intact FV functions in synergy with protein S as cofactor to APC [19], [20]. Platelets contain around 2.5% of the total protein S in blood, and it is released upon platelet activation [21]. The functional importance of protein S and protein C is evident from the increased risk of venous thrombosis affecting individuals with heterozygous deficiency of either protein [22].

Normal plasma contains 0.5-2.8 × 10⁶ MPs/mL [23], [24], but in several diseases, such as cancer [25], rheumatoid conditions [26], diabetes [27], systemic lupus erythematosus [24], atherosclerosis and coronary disorders [28], increased numbers of circulating MPs have been reported. Interestingly, an increased risk of thrombosis is observed in many of these diseases and it has been postulated that the circulating MPs contribute to the thrombosis risk.

While the procoagulant functions of platelet-derived MPs have been extensively investigated, few reports focus on the anticoagulant properties of MPs. Several studies have shown that APC-mediated inactivation of FVa on activated platelets is hampered and that platelet-derived FVa is less susceptible to APC-mediated degradation than plasma-derived FVa [29], [30], [31]. Degradation of FVa by APC on the surface of ionophore-activated platelets (MPs) has been shown to be more efficient than on platelets activated by thrombin, however compared to phospholipid vesicles, the degradation rate was still low [5], [32]. The APC-mediated degradation of FVIIIa on MPs has not been studied, but platelet-derived MPs can bind FVIIIa and FIXa [6], [7].

It has previously been shown that free, but not C4BP-bound, protein S specifically binds to platelet-derived MPs but not to resting or activated platelets [33]. To investigate whether the binding of protein S to the MPs renders them less procoagulant, the ability of protein S and FV to function as cofactors to APC on MPs was investigated.