Platelet activation in thrombotic disorders.

Recent advances have resulted in the elucidation of the principal molecular pathways of platelet function. Parallel studies have led to the identification of glycoprotein antigens whose presence at the platelet surface indicates an activated state. Such markers include GMP-140 and other glycoproteins of intracellular membranes whose translocation requires secretion and fusion of granule membranes with those joined to the surface.

Studies on the HLA Class-II Antigens of a Patient Presenting a Double Alloimmunization Following Posttransfusion Purpura.

In the Caucasian population, platelet incompatibility within the HPA-1 (Pl(A1/A2)) and HPA-5 (Br(a/b)) alloantigen systems are the two most likely causes of post-transfusion purpura (PTP) and neonatal alloimmune thrombocytopenia. However, the way in which HLA (class-II) antigens participate in alloantibody formation is unclear. The patient (M-J.

A defect of platelet aggregation associated with an abnormal distribution of glycoprotein IIb-IIIa complexes within the platelet: the cause of a lifelong bleeding disorder.

A young Italian man (A.P.) has a lifelong history of bleeding from gums and mucocutaneous tissue.

von Willebrand factor bound to glycoprotein Ib is cleared from the platelet surface after platelet activation by thrombin.

We recently reported that after activation of human platelets by thrombin, glycoprotein (GP) Ib-IX complexes are translocated to the surface-connected canalicular system (SCCS) (Blood 76:1503, 1990). As GPIb is a major receptor for von Willebrand factor (vWF) in platelet adhesion, we have now examined the consequences of thrombin activation on the organization of vWF bound to GPIb on the platelet surface. Studies were performed using monoclonal or polyclonal antibodies in either immunogold staining and electron microscopy (Au-EM) or in flow cytometry.

Further evidence that heparin-dependent thrombocytopenia may result from Fc receptor-mediated interactions.

Heparin-dependent thrombocytopenia (HDT) and associated thrombotic complications, are thought to be linked to the appearance of anti-platelet antibodies. Attempts were made to characterize the antibodies in the sera from 10 such patients. Western blotting against platelet antigens was inconclusive, often revealing multiple bands but with considerable variability from patient to patient.

Thrombin-induced platelet aggregates have a dynamic structure. Time-dependent redistribution of glycoprotein IIb-IIIa complexes and secreted adhesive proteins.

The role of glycoprotein (GP) IIb-IIIa complexes and of adhesive proteins in mediating platelet aggregation is now well defined. However, less is known of the changes that occur once aggregation has begun. We report immunogold staining of thin sections of platelets or platelet aggregates, embedded in Lowicryl K4M, after the use of polyclonal antibodies to GP IIb or GP IIIa, fibrinogen (Fg), von Willebrand factor (vWF), and thrombospondin (TSP).

Studies on the megakaryocytes of a patient with the Bernard-Soulier syndrome.

We have used monoclonal antibodies AP-1 (anti-GP Ib alpha). AP-2 (anti-GP IIb-IIIa) and FMC 25 (anti-GP IX) in immunofluorescence and immunocytochemical studies on megakaryocytes (MK) isolated from a Bernard-Soulier syndrome (BSS) patient whose giant platelets were characteristically deficient in GP Ib-IX complexes. Electron microscopy showed that the patient's MK were similar in size to normal MK.

Thrombin induces a rapid redistribution of glycoprotein Ib-IX complexes within the membrane systems of activated human platelets.

Previous studies have shown a decreased binding of monoclonal antibodies (MoAbs) to glycoprotein (GP) Ib-IX complexes on thrombin-stimulated platelets, but the reason for this is poorly understood. We have used (1) immunofluorescence procedures and flow cytometry, and (2) immunogold staining and electron microscopy to investigate this phenomenon. Washed platelets were incubated with alpha-thrombin, adenosine diphosphate, or ionophore A23187 for increasing lengths of time.

Megakaryocytes from the marrow of a patient with Glanzmann's thrombasthenia lacked GP IIb-IIIa complexes.

Although it is recognized that glycoprotein (GP) IIb-IIIa complexes are deficient in platelets in Glanzmann's thrombasthenia, little is known of the origin of the defect. We have examined the megakaryocytes in a bone marrow aspirate obtained from a thrombasthenia patient during surgery. Analysis of platelet proteins by SDS-polyacrylamide gel electrophoresis confirmed the patient to be of the type I subgroup.

Fibrinogen synthesis by megakaryocyte rich human marrow cell concentrates.

A rapid method is described for the production of a human marrow cell suspension highly enriched in megakaryocytes. These concentrates were incubated with radiolabelled amino-acids, and cell lysates were then analysed for fibrinogen synthesis. Neosynthesized proteins were detected by immunoprecipitation, immuno-affinity chromatography and electrophoresis.

Immunocytochemical study of the binding of fibrinogen and thrombospondin to ADP- and thrombin-stimulated human platelets.

We have used immunogold staining to locate thrombospondin (TSP) on thrombin-activated human platelets, and have compared its distribution with that of fibrinogen (or fibrin) on thrombin- and ADP-stimulated platelets. To do this, isolated platelets were incubated with monospecific antibodies to TSP or fibrinogen (fib) and the bound IgG located with a second antibody adsorbed to gold particles. Thrombin-induced secretion in Tyrode-Ca2+ was followed by both anti-TSP and anti-fib binding, with large clusters of gold particles observed on the platelet surface.

Distribution of glycoprotein IIb-IIIa complexes in the surface membranes of human platelets and megakaryocytes.

The distribution of glycoprotein (GP) IIb-IIIa complexes in the surface membranes of human platelets and megakaryocytes was investigated by transmission electron microscopy of cells that had been incubated with Fab fragments of a human alloantibody (IgG L) specific for the complex. Binding was visualized by a second antibody conjugated to peroxidase or adsorbed onto gold particles. Initial studies showed that the peroxidase reaction product and the gold particles were to be found at the outer surface of unactivated platelets.

Mepacrine labelling test and uranaffin cytochemical reaction in human megakaryocytes.

5-HT storage organelles were observed by electron microscope analysis in human megakaryocytes. They were less numerous per unit of surface than in platelets. Their number depended on the visualization technique employed.

Protein synthesis in human platelets correlation with platelet size.

Protein synthesis was investigated in human platelets by measuring incorporation of radio actively labelled amino acids into trichloroacetic acid (TCA) precipitable material. Platelet polysomes were characterized by their sedimentation rate in a sucrose gradient. It was confirmed that platelets synthesize proteins in their cytoplasm and that a part of their polysomes are bound to the skeletal framework.

Platelet dense bodies loaded with mepacrine. Study in chronic idiopathic thrombocytopenic purpura (ITP).

In 22 cases of chronic ITP, the platelet 5-HT storage organelles were counted by examination of platelets loaded with mepacrine and correlated with the size and volume of the platelets. Statistical analysis showed that the mean volume and the number of granules increased in ITP without increase in the mean number of granules per unit volume. A strong correlation was found between platelet long diameter and number of dense bodies in controls (44 healthy subjects) (r = 0.