Effect of Sulfhydryl Inhibitors on Platelet Agglutinability.

Abstract

Thrombin and adenosine diphosphate (ADP) induce platelet agglutination in the presence of appropriate divalent cations( 1, 2). The means by which these substances induce agglutination, and the relationship of agglutination to alterations in enzymatic activities of platelets are controversial. Through the use of sulfhydryl reagents and inhibitors, intact thiol groups have been demonstrated to be essential for maintenance of platelet integrity(3,4) and for certain enzymatic and biological functions of platelets( 5,6,7). However, the role of thiol groups in platelet agglutination is not clear. Grette found no inhibition of platelet viscous metamorphosis with the mexaptide forming agent Mersalyl at 35°C. With lower temperatures, a delay in the appearance of platelet aggregation was observed with Mersalyl ( 7). Zucker and Borrelli reported that mercuric chloride inhibited the thrombin-produced viscous metamorphosis of washed platelets (8). In platelet-rich plasma, platelet aggregation induced by thrombin or ADP was inhibited by 2.5 X 103 M mercuric chloride, but not by 2.5 X 10" M mercuric chloride or the thiol alkylating agent iodoacetate (9). The present study describes alterations in the ability of platelets to agglutinate with thrombin and ADP, in the presence of calcium, when platelets are incubated with certain sulfhydryl inhibitors.Materials and methods. Adenosine diphosphate, N-ethyl maleimide (NEM), and the sodium salt of p-chloromercuribenzoic acid (PCMB) were acquired from Mann Research Laboratories. Methyl mercuric nitrate (MMN) was prepared from recrystallized methyl mercuric iodide (Columbia Organic Chemicals Co.) by equimolar reaction with silver nitrate in methanol. The silver iodide which formed was removed by filtration and the product was crystallized twice from * This investigation was supported in part by a research grant from Nat. Inst. Health, P.H.S. methanol. Tris buffer, 0.05 M tris-hydroxymethyl aminomethane, pH 7.4, was made isotonic with sodium chloride. I t was used to prepare all inhibitor solutions and for making 22 mM solutions of CaClz and MgC12 from 0.22 M aqueous cation solutions. The solvent for ADP and thrombin was 0.154 M NaC1. Topical thrombin (Parke Davis & Co.) was partially purified on a G-25 Sephadex column equilibrated with normal saline. Thrombin unitage is expressed in Iowa units ( 10). Standard suspensions of washed canine platelets were prepared at 4OC from canine plasma collected in isotonic, pH 7.4, ethylenediamine tetra-acetic acid ( 1 1 ) . The washed platelets were suspended at a concentration of 400,00O/cmm in a solution of 0.0054 M sodium citrate and 0.146 M NaCl at 4OC. Platelet suspensions were kept in an ice bath and used within 4 hours of preparation. Only platelet preparations which gave 4+ agglutination with both thrombin (1.0 U / d , final concentration) and ADP were used. All glassware was silicone-coated (G. E. Dri-Film SC-87) except the test tubes in which agglutination tests were performed.The macroscopic platelet agglutination test of Brinkhous et aL(1...

The interaction of bovine factor VIII with human platelets.

“…Thiol groups are necessary for the aggregation of platelets by ADPR and thrombin and are readily blocked by NEM and PHMBS (33). Since NEM and PHMBS do not block Factor VIJI-induced aggregation, it seems unlikely that the thiol groups involved in ADP-and thrombin-induced aggregation are also involved in Factor VIII-induced aggregation.…”

“…Thiol groups on the platelet surface are essential for platelet aggregation by ADP and thrombin (33). Fig.…”

Scanning Electron Microscopic Observations on Human Blood Platelets and Their Alterations Induced by Thrombin

“…A divalent cation is not essential for thrombin-fibrinogen reaction, but essential, as a cofactor, for the induction of aggregation (SHERMER et al 1960 andROBINSON, MASON and WAGNER 1963). The ADP released from platelets by thrombin also causes their aggregation (GAARDER et al, 1961;BORN, 1962;BORN and CROSS, 1963;MAC-MILLAN, 1966).…”

Effect of Heparin on Platelet Adhesiveness

“…Platelets contain reactive SH-groups [17] which are important for platelet adhesiveness and aggregation [24,45]. Blockage of these groups inhibited platelet adhesiveness [53] and neither ADP nor heparin could overcome this effect.…”