thromboxane-b2 and Purpura--Thrombocytopenic--Idiopathic

Previous studies suggest that cyclooxygenase-2 (COX-2) might influence megakaryocyte (MK) maturation and platelet production in vitro. Using a gene deletion model, we analysed the effect of COX-2 deficiency on megakaryopoiesis and platelet function. COX-2-/- mice (10-12 weeks old) have hyper-responsive platelets as suggested by their enhanced aggregation, TXA2 biosynthesis, CD62P and CD41/CD61 expression, platelet-fibrinogen binding, and increased thromboembolic death after collagen/epinephrine injection compared to wild-type (WT). Moreover, increased platelet COX-1 expression and reticulated platelet fraction were observed in COX-2-/- mice while platelet count was similar to WT. MKs were significantly reduced in COX-2-/- bone marrows (BMs), with high nuclear/cytoplasmic ratios, low ploidy and poor expression of lineage markers of maturation (CD42d, CD49b). However, MKs were significantly increased in COX-2-/- spleens, with features of MK maturation markers which were not observed in MKs of WT spleens. Interestingly, the expression of COX-1, prostacyclin and PGE2 synthases and prostanoid pattern were modified in BMs and spleens of COX-2-/- mice. Moreover, COX-2 ablation reduced the percentage of CD49b+ cells, the platelet formation and the haematopoietic stem cells in bone marrow and increased their accumulation in the spleen. Splenectomy decreased peripheral platelet number, reverted their hyper-responsive phenotype and protected COX-2-/- mice from thromboembolism. Interestingly, fibrosis was observed in spleens of old COX-2-/- mice (28 weeks old). In conclusion, COX-2 deletion delays BM megakaryopoiesis promoting a compensatory splenic MK hyperplasia, with a release of hyper-responsive platelets and increased thrombogenicity in vivo. COX-2 seems to contribute to physiological MK maturation and pro-platelet formation.

Topics: Animals; Antigens, CD; Antigens, Differentiation; Blood Platelets; Bone Marrow; Crosses, Genetic; Cyclooxygenase 1; Cyclooxygenase 2; Hematopoietic Stem Cells; Hyperplasia; Megakaryocytes; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Platelet Count; Ploidies; Purpura, Thrombocytopenic, Idiopathic; Receptors, Thromboxane A2, Prostaglandin H2; Spleen; Splenectomy; Thromboembolism; Thrombophilia; Thrombopoiesis; Thromboxane B2

The blood volumes and concentrations of thromboxane B2 (TxB2), platelet factor 4 (PF4), and fibrinopeptide A (FPA) were measured every 30 seconds in bleeding-time blood in normal subjects and in patients with idiopathic thrombocytopenic purpura (ITP), delta and alpha delta storage pool deficiency (SPD), Bernard-Soulier Syndrome (BSS), thrombasthenia (TSA), and von Willebrand's disease (vWD). Data were fitted to second-order (TxB2, PF4, and FPA) or third-order (volumes) polynomials. Average values for various parameters over fixed-time intervals were determined by numerical methods. The bleeding time was greater than 15 minutes in all patient groups and the initial bleeding, as reflected by the initial slope of the fitted blood volume curves, was increased in ITP, BSS, and SPD (delta-SPD in particular), but not in vWD and TSA. The increased values for both the initial slope and the volume of blood collected after 2 minutes in SPD suggest that vascular tone may be modulated, in part, by dense granule substances such as adenosine triphosphate (ATP) or serotonin. In TSA, uniquely, both platelet (TxB2 and PF4) and coagulation (FPA) values were increased in early bleeding samples (initial slope). In vitro studies of TxB2 production, together with previous flow studies of fibrin formation, also suggest enhanced activation and coagulant properties of thrombasthenic platelets. In other patients, reduced values of all substances at later times may reflect impaired platelet-fibrin plug formation in the high-shear regions at the ends of transected blood vessels. However, the initial slopes of the fitted curves for both TxB2 and PF4 were normal in vWD, suggesting that the early appearance of these substances may typically be from platelets that are adherent to collagen within the lower shear environment of the wound surface. The finding that FPA values were not decreased initially in any patient group, including ITP, but were decreased at later times (except for TSA), suggests that early fibrin formation occurs independently of platelets in the low-shear environment of the wound surface, whereas at later times fibrin is formed in a platelet-dependent manner in the high-shear regions at the ends of transected vessels.

Topics: Adult; Bernard-Soulier Syndrome; Bleeding Time; Blood Platelet Disorders; Fibrinopeptide A; Humans; Kinetics; Middle Aged; Platelet Factor 4; Platelet Membrane Glycoproteins; Platelet Storage Pool Deficiency; Purpura, Thrombocytopenic, Idiopathic; Thromboxane B2; von Willebrand Diseases

Recently, we described a platelet antibody against a putative collagen receptor (P62), which was found in a patient with idiopathic thrombocytopenic purpura (ITP) (Blood 69:1712). We now report a deficiency of the P62 receptor in a young man whose platelets showed defective collagen-induced platelet aggregation. He had a mild bleeding tendency and slight thrombocytopenia. The results of coagulation and fibrinolysis studies were normal. The patient's platelets were partially unresponsive to collagen, although aggregation in response to ADP, thrombin, ristocetin, and calcium ionophore (A23187) was almost normal. Adhesion of his platelets to bovine collagen was markedly reduced. Addition of collagen caused no synthesis of thromboxane (TX)B2 in platelet rich plasma (PRP) from this patient. Furthermore, collagen produced no rise of cytosolic free calcium ([Ca2+]i) in fura2-loaded platelets. In contrast, thrombin caused TXB2 formation and an increase of [Ca2+]i in his platelets. These results suggest defective interaction between the platelets and collagen. The IgG from the ITP-patient induced irreversible aggregation in normal PRP, but caused no aggregation of the young man's platelets. Immunoblot studies showed that normal platelets had antigens with a molecular weight of 62 KDa under reducing conditions and of 57 KDa under nonreducing conditions. In contrast, the young man's platelets had no P62 band, although GPIa/IIa and thrombospondin were normally present. These results indicate that impaired collagen-induced aggregation in the patient's platelets was due to a deficiency of P62 and confirm that P62 may play a crucial role as a collagen receptor in platelet activation.

Topics: Adenosine Triphosphate; Adult; Blood Platelets; Calcium; Collagen; Electrophoresis, Gel, Two-Dimensional; Humans; Immunoblotting; Immunoglobulin G; Lectins; Male; Platelet Aggregation; Platelet Membrane Glycoproteins; Purpura, Thrombocytopenic, Idiopathic; Receptors, Cell Surface; Receptors, Collagen; Thrombospondins; Thromboxane B2