Anti-CD36 autoantibodies in thrombotic thrombocytopenic purpura and other thrombotic disorders: identification of an 85 kD form of CD36 as a target antigen.

The presence of anti-CD36 antibodies in plasma of patients with thrombotic thrombocytopenic purpura (TTP), idiopathic thrombocytopenic purpura (ITP), and heparin-induced thrombocytopenia without/with thrombosis (HIT/HITT) has been examined by immunoblots, and a monoclonal antibody capture assay, the platelet-associated IgG characterization assay (PAICA). Results with PAICA showed that 73% (8/11) of patients with TTP were positive, and 71% (10/14) by immunoblots. With ITP, 20% (6/30) were positive by PAICA and 19% (3/16) by immunoblots; HIT, 30% (3/10) were positive by PAICA and 60% (6/10) by immunoblot; HITT, 50% (2/4) by PAICA and 100% (4/4) by immunoblot. Purification of CD36 by fast protein liquid chromatography (FPLC) from Triton X-100 extracts of normal platelet membranes resulted in the isolation of two different forms: the classic 88 kD form, and a second, lighter 85 kD form. Our data indicated that the patients' plasma autoantibodies reacted strongly with the 85 kD form. Conventional monoclonal and polyclonal antisera produced to the 88 kD form reacted strongly with the 88 kD form but weakly with the 85 kD form. These results confirm the possible importance of anti-CD36 antibodies in the pathophysiology of TTP and other thrombocytopenias and demonstrate the presence of a previously unrecognized target antigen for these antibodies.

Cholesteryl ester accumulation in macrophages treated with oxidized low density lipoprotein.

The ability of CuSO4- and hypochlorite-oxidized LDL to promote cholesterol accumulation in macrophages was examined. Both CuSO4- and hypochlorite-oxidized LDL were rapidly metabolized by mouse peritoneal macrophages to a level approximately 10 times that observed for native LDL and both modified lipoproteins increased the accumulation of unesterified cholesterol. However when each modified lipoprotein was incubated with macrophages for 40h, only hypochlorite-oxidized LDL produced significant accumulation of cholesteryl esters, with levels approaching 85 micrograms/mg cell protein. This finding was verified by nile red staining. The cholesteryl ester content of cupric sulfate-modified LDL was found to be significantly decreased when compared to either native or hypochlorite-modified LDL promotes massive cholesteryl ester accumulation because the cholesteryl ester content of the LDL particle is preserved.

Advances in agarose gel electrophoresis of serum lipoproteins.

Agarose gel electrophoresis has been extensively employed by researchers to gain a greater understanding of lipoprotein biology and its relationship to cardiovascular disease. Advances in this technique have been made in the visualization and quantitation of separated lipoproteins, in the use of agarose gel electrophoresis for detection and quantitation of apolipoproteins of the separated lipoproteins, and in the detection of lipoprotein heterogeneity. Agarose gel electrophoresis has been employed for two-dimensional electrophoretic analysis of lipoproteins as well as in several different methods which probe the immunological properties of lipoproteins. Agarose gel electrophoresis has thus become an important tool in the study of serum lipoproteins in both clinical and basic science laboratories.

Effects of phosphatidylserine on the oxidation of low density lipoprotein.

1. LDL was incubated in the presence of 1 microM CuSO4 for 18 hr at 37 degrees C. The content of lipoperoxides was found to be approx. 40 nmol MDA equivalents/mg LDL protein. The addition of 50 microM phosphatidylserine (PS) reduced the content of lipoperoxides to 15% of control values. 2. The electrophoretic mobility observed for LDL oxidized in the presence of PS approximated the mobility observed for native LDL. 3. The formation of conjugated dienes was strongly inhibited when LDL was oxidized in the presence of PS. 4. The addition of 50 microM phosphatidylcholine, phosphatidylglycerol and cardiolipin did not alter the extent of LDL oxidation. 5. PS did not inhibit the oxidation of LDL mediated by J774 macrophages in the presence of Ham's F-10 culture medium. Under these conditions, PS was found to be an excellent substrate for oxidation.

The minimum surface fibrinogen concentration necessary for platelet activation on dimethyldichlorosilane-coated glass.

Albumin and fibrinogen were competitively adsorbed onto dimethyldichlorosilane-coated glass (DDS-glass) and platelet activation was examined as a function of the surface fibrinogen concentration. The weight ratio of albumin to fibrinogen in the adsorption solution was varied from 10 to 700. Platelet activation was quantitated by the area and circularity of spread platelets. When the DDS-glass was coated with albumin alone, platelets were only contact adherent and could not spread at all. After competitive adsorption of fibrinogen and albumin, however, platelets were able to spread on the surface. Platelet activation increased linearly as the surface fibrinogen concentration increased up to 0.02 micrograms/cm2. Platelets were able to activate fully if the surface fibrinogen concentration was 0.02 micrograms/cm2 or higher, even though the surface was dominated by albumin. It appears that platelets can activate fully as long as only a small fraction (2-15%) of the surface is covered with tightly bound fibrinogen.

Morphological characterization of surface-induced platelet activation.

Morphological changes of platelets activated on glass and dimethyldichlorosilane-treated glass were investigated using video microscopy. The platelet morphological changes were quantified by measuring the area and circularity of spreading platelets. In addition, re-organization of cytoskeletal structures of spread platelets was examined. The effects of precoated albumin and fibrinogen on the platelet spreading kinetics were examined as a function of surface protein concentrations. Results showed that platelet shape changes were very sensitive to the surface concentration of precoated proteins. In general, platelets on fibrinogen-precoated surfaces spread fully to a circular shape and developed an extensive inner filamentous zone. In the presence of albumin on the surface, however, platelets could not spread fully and the development of the inner filamentous zone was very poor. For both albumin and fibrinogen, the maximum effects of precoated proteins on platelet shape changes were observed when the surface protein concentration reached the monolayer concentration.