Novel platelet-agglutinating protein from a thrombotic thrombocytopenic purpura plasma.

Abstract

A novel platelet-agglutinating protein (PAP) was purified -2,000-fold from the plasma of a patient with thrombotic thrombocytopenic purpura (TTP) by ammonium sulfate fractionation, DEAE-Sephacel and concanavalin A-Sepharose chromatographies. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with and without reduction, this preparation revealed a major protein band with a molecular weight of 37,000, and a minor band with a molecular weight of 32,000-34,000. After elution from the gel, only the 37,000-mol wt protein corresponding to the major band induced the platelet agglutination. When four normal plasmas and the recovery plasma from the same TTP patient were subjected to the similar purification steps, the 37,000-mol wt major band was absent. The 125I-PAP bound to the platelets in a concentration-dependent manner. The platelet agglutination induced by PAP was not inhibited by hirudin, heparin in the presence of antithrombin III, phenylmethylsulfonyl fluoride, apyrase, aspirin, or prostaglandin I2. However, it was inhibited by IgG from normal adults and from the same TTP patient after recovery.The anti-37,000-mol wt PAP antiserum prepared in the rabbit formed a single precipitin line against the highly purified PAP. Using this antiserum in the Western immunoblotting, the 37,000-mol wt protein band was found in the three TTP plasmas, of which the platelet-agglutinating activity was inhibited by the anti-37,000-mol wt PAP IgG. The 37,000-mol wt immunoprecipitin band was absent in the plasmas obtained from another two TTP patients, two normal subjects, two patients with idiopathic thrombocytopenic purpura, and two patients with disseminated intravascular coagulation. These results suggest that the 37,000-mol wt PAP is present only in certain cases of T1P, and is likely to be responsible for the formation of platelet thrombi in the microcirculation.

Platelet-Agglutinating Protein p37 from a Patient with Thrombotic Thrombocytopenic Purpura Has Characteristics Similar to Prethrombin 2

“…This was similar to most of the chemical characteristics of PAP p37 that are described by Siddiqui and Lian (Siddiqui and Lian, 1985, 1993, except for the weak binding activity to concanavalin A (Con-A). PAP p37 has a molecular weight of 37 kDa, as well as single polypeptide of acid glycoprotein with potent plateletagglutinating activity.…”

“…Recent suggestions are that persisting-endothelial-derived multimeric von Willebrand factor (vWF), or unusually large vWF, which cannot be cleaved normally (Wagner et al, 1995;Tsai and Lian, 1998;Veyradier et al, 2001), and 37 kDa plateletagglutinating protein (PAP), p37, are associated with these pathologic mechanisms (Lian et al, 1979(Lian et al, , 1991Siddiqui and Lian, 1985, 1993Jin et al, 1995;Chen S and Lian 1998). Other investigators reported that there were reduced prostacyclin I 2 (PG I 2 ) production (Wu KK, 1992), or *To whom correspondence should be addressed.…”

“…These are associated with the deficiency of the vWF-cleaving protease, or the formation of an antibody against the protease (Tsai and Lian, 1998;Tsai et al, 2000). On the other hand, PAP p37 was found in the plasma of TTP patients, which are known to have potent platelet-agglutination activity that was first described by Siddiqui and Lian (Siddiqui and Lian, 1985).…”

The pathophysiology and management of thrombotic thrombocytopenic purpura

“…The same authors identified a 37 kD protein (p37) with a platelet agglutinating activity that was independent from ADP release, energy metabolism and the cyclooxygenase pathway. In turn, p37 was completely inhibited by IgG from healthy human adults or from TTP patients after recovery through the formation of a p37-IgG complex (61). p37 activity appears mediated by a specific and concentrationdependent binding to a 97 kD platelet membrane protein.…”

Effect of plasma exchange on cytokines measured by multianalyte bead array in thrombotic thrombocytopenic purpura

“…Endothelial cell injury, platelet aggregation, and abnormal von Willebrand factor (vWF) homeostasis have been implicated in its pathogenesis [6][7][8][9][10][11][12][13][14][15][16][17].…”