Binding of Platelet Agglutinating Protein p 37 from the Plasma of a Patient with Thrombotic Thrombocytopenic Purpura to Human Platelets

Abstract

SummaryWe have previously reported the purification of a 37 kDa platelet agglutinating protein (PAP p37) from the plasma of a patient with thrombotic thrombocytopenic purpura (TTP) that was shown to be present in a subset of TTP patients. To gain further insight into the interaction between PAP p37 and platelets, we have studied the properties of PAP p37 binding to platelets. Washed human platelets from two normal donors and two TTP patients after recovery were used for the experiments. The PAP p 37 binding to platelets was specific, concentration dependent and saturable. Scatchard analysis demonstrated about 20,564–27,090 PAP p37 binding sites per platelet. Stimulation of platelets with thrombin or ADP did not have any significant effect on its binding.Thiol- and serine-specific protease inhibitors did not inhibit PAP p37 binding to the platelets. Sugars such as glucose, fructose, mannose, and sialic acid, at 40 mM, inhibited its binding to platelets by 44%, 73%, 79%, and 91% respectively, but galactose and amino sugars did not have any significant effect. At 250 μg/ml, Concanavalin-A inhibited 42% of binding, but other lectins, such as phytohemagglutinin-P, potato lectin and helix pomatia lectin (snail), did not. Pre-incubation of 125I-PAP p37 with the adult human IgG, decreased its binding to the platelets. The monoclonal antibodies to GP Ib (6D1) and GP IIb-IIIa complex (10E5) did not inhibit the binding of 125I-PAP p37 to platelets. Fibrinogen and von Willebrand factor did not affect the binding either. These results suggest that PAP p37 binds to platelets on the sites other than GP lb or GP IIb-IIIa complex.

Minimal evidence of platelet and endothelial cell reactive antibodies in thrombotic thrombocytopenic purpura

“…However, it is unclear whether various investigators identified the same entity. For example, Lian reported a platelet aggregating factor that was inhibited by human IgG [20,21], whereas the aggregating factor reported by Kelton was unaffected by IgG [24,25]. Moreover, platelet aggregating activity and other TTP-associated abnormalities have been detected in plasma from patients with a variety of other diagnoses, suggesting that the phenomena may not be specific to TTP [22,23,26,27].…”

“…The implicated factor has been identified as a 37 kDa platelet agglutinating protein (PAP p37) that has been reported to bind to platelet GP IV [20,21]. Other investigators observed similar platelet aggregating activity in TTP plasma or serum [3,[22][23][24].…”

Platelet activation in patients with thrombotic thrombocytopenic purpura

“…Alternatively, TTP patients may be deficient in some protective component found in normal plasma. Platelet agglutinating activity [4,5] and platelet activating factors [6] have been detected in the plasma of subsets of TTP patients.…”

History of Thrombotic Thrombocytopenic Purpura and the von Willebrand Factor–Cleaving Protease, ADAMTS13

“…A putative plasma factor stimulating the endothelial cell synthesis and release of prostacyclin (prostaglandin I 2 ), a potent inhibitor of platelet aggregation, was suggested to be missing in patients with hemolytic uremic syndrome (HUS) and TTP [ 11 ]. A 37 kDa protein agglutinating normal platelets [ 12 ] or a 59 kDa protein aggregating platelets from healthy donors in the presence of Ca ++ and fi brinogen [ 13 ] were isolated from the plasma of a few patients with acute TTP. A calcium-dependent cysteine protease was found in the sera of 15 patients during an acute bout of TTP but not during remission and was identifi ed to activate platelets [ 14 ].…”