New insights into the role of erythrocytes in thrombus formation.

The role of erythrocytes in thrombus formation has previously been regarded as passive by their influence on rheology. Erythrocytes are known, due to their abundance and size, to push platelets to the vascular wall (laminar shearing). This results in an increased platelet delivery at the vascular wall enabling platelets to seal off a vascular damage preventing excessive blood loss. Recently, there is new evidence for erythrocytes to influence thrombus formation in multiple ways besides their effect on rheology. Several groups have shown that besides platelets, erythrocytes are the main suppliers of phosphatidylserine-exposing membranes needed for coagulation resulting in fibrin formation. In addition, our group has found that the intercellular adhesion molecule 4-αIIbß3 interaction mediates erythrocyte-platelet interaction in flowing blood. By inhibiting this interaction, we found decreased thrombin formation and decreased incorporation of erythrocytes into a thrombus. This review will provide more in-detail information of existing and new hypotheses regarding the role of erythrocytes in thrombus formation.

From antibody to clinical phenotype, the black box of the antiphospholipid syndrome: pathogenic mechanisms of the antiphospholipid syndrome.

The antiphospholipid syndrome (APS) is diagnosed by the combination of vascular thrombosis and/or pregnancy morbidity and the detection of antiphospholipid antibodies (aPLs) in plasma. In the last few years, a great effort has been made to unravel the mechanism by which aPLs cause thrombosis and a vast amount of mechanisms have been proposed. aPLs were proposed to induce a prothrombotic state by influencing the cellular blood compartment, the plasma compartment, the vascular wall and even metabolic pathways beyond the hemostatic system. However, due to the diversity in the mechanisms and the differences in the methodology, the focus of the mechanistical studies in this field seems to be largely diffused. It is hard to imagine that aPLs can exert such a diversity of effects, resulting in either thrombosis and/or pregnancy morbidity and the relationship between aPLs and the clinical manifestations remains to be a mysterious "black box". In an attempt to get insight in what takes place inside the black box, we have analyzed 126 mechanistical studies on aPLs and discussed differences in the type of antibodies that were used, the involvement of beta2-glycoprotein I (ß2GPI), and the criteria used to diagnose APS patients.

Accelerated uptake of VWF/platelet complexes in macrophages contributes to VWD type 2B-associated thrombocytopenia.

Von Willebrand disease (VWD) type 2B is characterized by mutations causing enhanced binding of von Willebrand factor (VWF) to platelets. Bleeding tendency is associated with heterogeneous clinical manifestations, including moderate to severe thrombocytopenia. The underlying mechanism of the thrombocytopenia has remained unclear. Here, a mouse model of VWD type 2B was used to investigate pathways contributing to thrombocytopenia. Immunohistochemical analysis of blood smears revealed that mutant VWF was exclusively detected on platelets of thrombocytopenic VWD type 2B mice, suggesting that thrombocytopenic VWD type 2B mice were elevated two- to threefold upon chemical macrophage depletion. Colocalization of platelets with CD68-positive Kupffer cells and CD168-positive marginal macrophages in liver and spleen, respectively, confirmed the involvement of macrophages in the removal of VWF/platelet complexes. Significantly more platelets were found in liver and spleen of VWD type 2B mice compared with control mice. Finally, platelet survival was significantly shorter in VWD type 2B mice compared with control mice, providing a rationale for lower platelet counts in VWD type 2B mice. In conclusion, our data indicate that VWF type 2B binds to platelets and that this is a signal for clearance by macrophages, which could contribute to the thrombocytopenia in patients with VWD type 2B.

Indications for a protective function of beta2-glycoprotein I in thrombotic thrombocytopenic purpura.

It has been shown that ß(2) -glycoprotein I (ß(2) GPI) interacts with von Willebrand factor (VWF) in a glycoprotein (GP)Ib binding state. Given the presence of active VWF multimers in thrombotic thrombocytopenic purpura (TTP), we speculated that ß(2) GPI might play a role in TTP. We found that ß(2) GPI plasma levels were significantly lower in acute and remission TTP patients than in normal controls, showing a direct correlation with ADAMTS 13 levels and an inverse correlation with the extent of VWF activation. In vitro flow experiments demonstrated that ß(2) GPI can block platelet adhesion to endothelial cell-derived VWF strings. We confirmed the direct binding of ß(2) GPI to VWF by surface plasmon resonance, and determined that domain I of ß(2) GPI is the binding site of VWF A1 domain. Adhesion of ß(2) GPI to erythrocytes and platelets was increased in the presence of active VWF, indicating that ß(2) GPI may be cleared from the circulation during TTP episodes together with blood cells. Our findings suggest that ß(2) GPI may protect from the effects of hyper-functional VWF by inhibiting its interaction with platelets.

Arachidonic acid depletion extends survival of cold-stored platelets by interfering with the [glycoprotein Ibα--14-3-3ζ] association.

BACKGROUND: Cold storage of platelets reduces bacterial growth and preserves their hemostatic properties better than current procedures do. However, storage at 0°C induces [14-3-3ζ-glycoprotein Ibα] association, 14-3-3ζ release from phospho-Bad, Bad activation and apoptosis. DESIGN AND METHODS: We investigated whether arachidonic acid, which also binds 14-3-3ζ, contributes to coldinduced apoptosis. RESULTS: Cold storage activated P38-mitogen-activated protein kinase and released arachidonic acid, which accumulated due to cold inactivation of cyclooxygenase-1/thromboxane synthase. Accumulated arachidonic acid released 14-3-3ζ from phospho-Bad and decreased the mitochondrial membrane potential, which are steps in the induction of apoptosis. Addition of arachidonic acid did the same and its depletion made platelets resistant to cold-induced apoptosis. Incubation with biotin-arachidonic acid revealed formation of an [arachidonic acid-14-3-3ζ-glycoprotein Ibα] complex. Indomethacin promoted complex formation by accumulating arachidonic acid and released 14-3-3ζ from cyclo-oxygenase-1. Arachidonic acid depletion prevented the cold-induced reduction of platelet survival in mice. CONCLUSIONS: We conclude that cold storage induced apoptosis through an [arachidonic acid-14-3-3ζ-glycoprotein Ibα] complex, which released 14-3-3ζ from Bad in an arachidonic acid-dependent manner. Although arachidonic acid depletion reduced agonist-induced thromboxane A(2) formation and aggregation, arachidonic acid repletion restored these functions, opening ways to reduce apoptosis during storage without compromising hemostatic functions post-transfusion.

The platelet P2Y12 receptor contributes to granule secretion through Ephrin A4 receptor.

The main responses of P2Y(1) ligation are platelet shape change and transient aggregation while P2Y(12) ligation amplifies P2Y(1)-induced aggregation and accelerates aggregation, secretion and thromboxane A(2) production induced by other agonist-receptor complexes. We searched for new targets of P2Y signalling using micro-arrays with 144 peptides representing known phosphosites of protein tyrosine kinases. ADP induced phosphorylation of peptides representing surface receptors, second messenger enzymes and cytoskeletal proteins. Strong phosphorylation was found in peptides representing Ephrin-receptor family members. Blockade of P2Y(1/12) inhibited phosphorylation of EphA4- and EphB1-peptides on micro-arrays. The EphA2/4 inhibitor 2,5-dimethylpyrrolyl benzoic acid derivative interfered with P2Y(1/12)-induced EphA4 phosphorylation, left P2Y(1)-induced aggregation unchanged but inhibited with P2Y(12)-induced secretion, second phase aggregation and thrombus formation on collagen at 1600 s(-1). These results show that platelet EphA4 is an important intermediate in P2Y(12)-induced granule secretion.

Platelet protein disulfide isomerase is localized in the dense tubular system and does not become surface expressed after activation.

Evidence is accumulating that circulating tissue factor (TF) contributes to the initiation of coagulation and the formation of fibrin. The majority of circulating TF is cryptic, and it has been suggested that close vicinity with anionic phospholipids on the cell surface increases the active conformation of TF. Two recent papers have shown that encryption of TF and initiation of coagulation are facilitated by the enzyme protein disulfide isomerase (PDI), possibly on the surface of activated platelets or endothelial cells. In this brief report, we demonstrate that the majority of PDI in platelets is intracellular where it is exclusively located in the dense tubular system. On activation, PDI remains confined to the intracellular stores of the dense tubular system and is neither released nor targeted to the cell surface. Similar results were obtained in endothelium where PDI remains exclusively localized in the endoplasmic reticulum, both at steady state and after thrombin stimulation.