G. A. Jamieson

Anti‐CD36 antibodies in thrombotic thrombocytopenic purpura

Summary. The membrane glycoprotein CD36 (GPIV, Mr 88000) is found on platelets, monocytes and endothelial cells of the microvasculature. In the present study, anti CD36 antibodies have been identified as occurring with high frequency in patients with thrombotic thrombocytopenic purpura. The presence of anti CD36 antibodies in 15 TTP plasma samples thought to contain them on the basis of an initial screening by protein blots was confirmed by re‐screening against a standard of purified CD36, by immunoprecipitation from 125I‐labelled control platelets and by dot blots against purified CD36. In a further 28 random samples examined, 23/27 (85%) were CD36‐positive by immunoprecipitation, 21/28 (75%) by protein blotting, and 17/28 (60%) by dot blots against purified CD36. On protein blots following SDS‐PAGE, immunoprecipitates produced from normal platelets by TTP plasma gave positive reactions with the anti CD36 monoclonal antibody 125I‐Mo91. One half of the total TTP samples examined (21/42) caused 70% release in control platelets loaded with 14C‐serotonin. Of samples causing release 70%, one‐half (8/15) failed to cause release from Naka‐negative platelets which constitutively lack CD36 showing that CD36 was the sole target for platelet activation in these TTP samples. These studies demonstrate that antibodies directed against CD36 occur frequently in TTP patients and could cause thrombotic complications and vascular damage by reacting with the parent antigen present in platelets and endothelial cells.

Inhibition of platelet adhesion to collagen by monoclonal anti‐CD36 antibodies

Monoclonal anti CD36 antibodies capable of inhibiting platelet adhesion to collagen have not previously been identified. We have now prepared two groups of monoclonal antibodies. One group was prepared using, as immunogen, highly purified (99+%) CD36 prepared by a denaturing procedure. These antibodies (Mo series) reacted strongly with CD36 on protein blots but did not immunoprecipitate native CD36 from platelet lysates nor inhibit platelet adhesion to collagen. The second group of monoclonal antibodies (131 series) was prepared using CD36 purified to >95% by a non‐denaturing procedure. These antibodies reacted with control platelets, but not Naka‐negative platelets which lack CD36, as measured by flow cytometry and by immunoprecipitation. Three monoclonal antibodies of this latter group (131.4, 131.5 and 131.7) inhibited platelet adhesion to collagen in static systems under Mg2+‐independent conditions but had little effect in the presence of Mg2+. 131.4 and 131.7 also inhibited adhesion to collagen using citrated whole blood in a parallel plate flow chamber at physiological shear rates (800 s−1), whereas 131.5 was without effect. These are the first anti‐CD36 monoclonal antibodies shown to be capable of inhibiting platelet adhesion to collagen and provide further evidence that CD36 plays a role in platelet–collagen interaction.

High and moderate affinity pathways for α-thrombin-induced platelet activation

Abstract. The interpretive review represents evidence for two pathways of α-thrombin-induced activation in human platelets and evaluates the possible roles of a functional thrombin receptor recently identified by expression cloning. [P.S.E.B.M. 1991, Vol 198]

Human CD36 deficiency is associated with elevation in low-density lipoprotein-cholesterol.

To find out whether CD36 plays a role in the human lipoprotein metabolism, we studied lipoprotein profiles in subjects with CD36 deficiency. Apparently healthy Japanese volunteers (n = 790) were classified by flow cytometry into three groups of normal (platelet and monocyte CD36+, n = 741, 93.8%), type-II deficiency (platelet CD36- and monocyte CD36+, n = 45, 5.7%), and type-I deficiency (platelet and monocyte CD36-, n = 4, 0.5%). At least one of reported mutations in the CD36 gene was found in all four subjects with type-I deficiency and in 23 of the 45 subjects with type II. Among 779 subjects (731 normals, 44 type II, and four type I) with serum triglyceride levels of <400 mg/dL, serum total cholesterol and low-density lipoprotein (LDL) cholesterol were significantly elevated in type-II deficiency (P = 0.0095 and 0.0382 versus normal, respectively, Scheffes F-test), while differences were not significant in triglyceride and high-density lipoprotein-cholesterol. Similar tendency was observed in type-I deficiency, although the differences were not statistically significant because of small sample size. We conclude that CD36 deficiency elevates LDL cholesterol, indicating a contribution of CD36 to LDL metabolism.

Anti‐CD36 antibodies in patients with lupus anticoagulant and thrombotic complications

Summary . Six patients with lupus anticoagulant with thrombotic complications, but not exhibiting systemic lupus erythematosus, demonstrated the presence in their plasma of antibodies directed against platelet antigens which were not detectable in two patients presenting with lupus anticoagulant but without thrombotic complications. Protein blotting of separated normal platelet proteins against patient plasma gave up to 18 bands of varying intensity indicative of multiple antiplatelet antibodies; one of these antibodies recognized a component with a mobility identical with CD36 (GPIV; m.w. 88,000) in 4/6 cases. Antibodies to CD36 and one or two other components were identified in 5/6 cases by immunoprecipitation from 125I‐labelled control platelets and 6/6 by dot blots against purified CD36. These results suggest that antiplatelet antibodies and, specifically, anti CD36 antibodies, occur frequently in the plasma of patients presenting with lupus anticoagulant and thrombotic complications.

Weak platelet agonists and U46619 induce apoptosis-like events in platelets, in the absence of phosphatidylserine exposure.

Platelets express apoptotic markers during storage, while aging and after stimulation with strong agonists thrombin and collagen. It is unknown if the weak agonists ADP and epinephrine or U46619, a thromboxane analog, induce the expression of apoptotic markers in platelets. To answer this question, we measured phosphatidylserine exposure, gelsolin cleavage and decrease in membrane mitochondrial potential after stimulation with these agonists. No phosphatidylserine exposure was evident, however, gelsolin cleavage and a platelet population with a decreased membrane mitochondrial potential appeared, suggesting that in platelets selective agonists can induce apoptosis in the absence of phosphatidylserine exposure. Interestingly, costimulation by thrombin plus collagen together with each of the other agonists increased the phosphatidylserine exposure induced by strong agonists. These findings may be of importance in platelet activation and apoptosis under pathophysiological conditions where multiple effectors are involved.

Antibodies to CD36 (GPIV) Inhibit Platelet Adhesion to Subendothelial Surfaces Under Flow Conditions

The membrane glycoprotein CD36 (glycoprotein [GP] IV) has previously been shown to accelerate the initial interaction of platelets with purified type I collagen in both static and flow systems. In the present study, the role of CD36 on platelet interaction with physiologically relevant collagenous surfaces was addressed. Using arterial subendothelium (SE) and endothelial cell extracellular matrix (ECM), studies were performed under flow conditions with annular and parallel-plate perfusion chambers, respectively, at a shear rate of 800 s-1 for 2, 5, and 10 minutes. Perfusates consisted of citrated normal blood samples incubated with Fab fragments of a monospecific polyclonal anti-CD36 antibody or with each of three new anti-CD36 monoclonal antibodies (MoAbs) that inhibit platelet adhesion to purified type I collagen in a static system (131.4, 131.5, and 131.7). Perfusions over SE were also carried out using citrated blood samples from a Naka-negative donor, whose platelets lack CD36. Morphometric evaluation of the perfused samples showed that polyclonal anti-CD36 Fab and the three monoclonal anti-CD36 antibodies inhibited platelet adhesion to the two substrates by 40% after 2 minutes of perfusion and by 30% after 5 minutes (P < .005 on SE and P < .01 on ECM), but at 10 minutes, significant inhibition was seen only on SE with polyclonal anti-CD36 Fab. Similar inhibitions were seen with Naka-negative platelets on SE. These studies demonstrate that CD36 plays a role in the early stages of platelet adhesion to physiologically relevant subendothelial surfaces.

Platelet Activation by α-Thrombin Is a Receptor-Mediated Eventa

Computer-assisted data analysis of binding isotherms (LIGAND) has shown that human platelets have binding sites for alpha-thrombin of high (Kd 0.3 nM), moderate (Kd 10 nM), and low affinities (Kd 3 microM). Application of similar techniques has shown that TLCK-thrombin does not, whereas PPACK-thrombin does, bind to the high-affinity binding site accessible to alpha-thrombin, but that both bind to the moderate and low-affinity sites. Treatment of platelets with Serratia marcescens protease destroys the high-affinity site but does not affect moderate-affinity binding. In accordance with this model, both modified thrombins compete with alpha-thrombin for platelet activation at the moderate-affinity site, but only PPACK-thrombin competes at the high-affinity site. These results establish that platelet activation by either low or moderate concentrations of thrombin are receptor-mediated events and explain the paradox of the differential effects of TLCK-thrombin on the binding and activation of platelets by alpha-thrombin.

Synergistic effects of lectins in the interaction of thrombin with human platelets.

Several lectins have been studied for their effects on the interaction of thrombin with human platelets. Wheat germ agglutinin, concanavalin A and Ricinus communis lectin increased the number of high affinity sites for diisopropylphosphothrombin on washed platelets from 3000 to about 12 000 but the binding affinities were unchanged (Kd approx 4 nM). Two other lectins, Lens culinaris and Bandieria simplicifolia, were without effect. (2) Using formalinized platelets to avoid possible complications of the platelet release reaction, wheat germ agglutinin showed a marked increase (5-fold) in the binding of active thrombin, peanut agglutinin had no effect while Ricinus communis and :Bandieria simplicifolia showed marginal increases (2-fold). Thrombin binding was decreased to about one quarter with Lens culinaris, Phaseolus vulgaris and concanavalin A. (3) Wheat germ agglutinin caused a synergistic increase of platelet aggregation at low concentrations of thrombin (12.5 mU/ml) and ADP (1 microM), both in the absence and presence of added fibrinogen, but had no effect on ristocetin-induced aggregation.

Efficient tyrosine phosphorylation of proteins after activation of platelets with thrombin depends on intact glycoprotein Ib.

The role of platelet glycoprotein Ib as a thrombin receptor has been often a subject of controversy. We have investigated the role of the thrombin receptors, GPIb and protease-activated receptor (PAR)-1. Tyrosine phosphorylation in whole platelet lysates and in cytoskeletal extracts was evaluated after activation with thrombin and with the thrombin receptor-activating peptide (TRAP). Different experimental approaches were applied including: (i) congenital deficiency of platelet GPIb (Bernard Soulier syndrome, BSS), (ii) antibody to GPIb (AP1), (iii) selective protease cleavage (metalloprotease), and (iv) antibody to (PAR)-1. After activation of control platelets with thrombin or TRAP, multiple proteins became tyrosine phosphorylated in platelet lysates and some of them associated with the cytoskeletal fraction. These effects were absent in BSS platelets. Presence of AP1 or metalloprotease treatment showed an inhibitory effect when platelets were activated with a low concentration of thrombin or TRAP. Blockade of PAR-1 with a specific antibody, SPAN 12, inhibited platelet response to both agonists. This study reinforces the hypothesis that GPIb is the high-affinity receptor for thrombin. The signaling mechanisms occurring through tyrosine phosphorylation of proteins triggered by thrombin seem to be dependent on intact GPIb. Moreover, our results indicate that both receptors, GPIb and PAR-1, are necessary to achieve a full platelet response to thrombin.