Rei Matsueda

New reagents for the introduction of the thiomethyl group at sulfhydryl residues of proteins with concomitant spectrophotometric titration of the sulfhydryls: Methyl 3-nitro-2-pyridyl disulfide and methyl 2-pyridyl disulfide

Abstract Two new reagents for the titration of sulfhydryl groups in peptides and proteins and for their temporary blocking with the thiomethyl group have been developed. The sulfhydryl groups in cysteine, glutathione, and papain react quantiatively under mild conditions with these reagents, methyl 3-nitro-2-pyridyl disulfide and methyl 2-pyridyl disulfide, with concomitant methanethiolation and without the need to employ a large excess of reagent. Because of the chromophoric properties of the 3-nitro-2-thiopyridone and 2-thiopyridone products, spectrophotometric titration of the sulfhydryl groups can be carried out, accompanying their methanethiolation. The modification of the sulfhydryl groups in peptides and proteins with thiomethyl is rapidly and completely reversible upon addition of thiols such as l -cysteine.

Aggregation of washed platelets by plasminogen and plasminogen activators is mediated by plasmin and is inhibited by a synthetic peptide disulfide

Plasmin is known to activate platelets. However, it is not clear whether plasminogen activators as used in thrombolytic therapy can aggregate platelets and how this relates to the ability of each activator to convert plasminogen to plasmin. Urokinase (UK) and streptokinase (SK) activated purified plasminogen (2 microM) in a concentration-dependent manner. The rates of aggregation of washed platelets by the above plasminogen activators and plasminogen were similar to the extent of activation of plasminogen to plasmin in the absence of platelets. UK or SK (0.2 microM) and plasminogen (2 microM) aggregated platelets modified by an ADP affinity analog, 5-p-fluorosulfonylbenzoyladenosine (FSBA), and cleaved aggregin, a putative ADP receptor, in [3H]FSBA-modified platelets. These results suggest that the effect was independent of ADP. In contrast, incubation mixtures containing only plasminogen (2 microM) and single chain tissue plasminogen activator (sc-tPA) (less than or equal to 0.12 microM) neither activated the zymogen to an appreciable extent nor aggregated platelets. But, in the presence of fibrin(ogen) fragments (tPA-stimulator), a mixture of plasminogen and sc-tPA aggregated unmodified and FSBA-modified platelets, and cleaved aggregin. The results imply that platelets, in the presence of t-PA stimulator, potentiate activation of plasminogen to plasmin by t-PA, as previously reported. P1, Phe-Gln-Val-Val-Cys-(NpyS)-Gly-NH2, (NpyS = 3-nitro-2-thiopyridine), a synthetic hexapeptide capable of binding to and inhibiting calpain, has been shown to inhibit platelet aggregation induced by purified plasmin. P1 inhibited platelet aggregation by plasminogen and any of the three plasminogen activators. Our results show that at plasma concentrations of plasminogen and at levels of UK and SK attained after infusion of these agents during thrombolysis, these mixtures can cause maximum aggregation which may contribute to reocclusion and stenosis following infarct therapy. P1 can effectively inhibit platelet aggregation under such conditions.

Specificity of the sequence in Phe-Gln-Val-Val-Cys (-3-nitro-2-pyridinesulfenyl)-Gly-NH2--a selective inhibitor of thrombin-induced platelet aggregation.

Thrombin-induced platelet aggregation is mediated in part by the intracellularly activated calpain expressed onto the external side of the membrane. We have previously shown that P1, Phe-Gln-Val-Val-Cys(Npys)-Gly-NH2 [Npys = 3-nitro-2-pyridinesulfenyl], an affinity analog corresponding to the highly conserved sequence Gln-Val-Val-Ala-Gly-NH2, present in domains 2 and 3 of human kininogens, was an irreversible inhibitor of platelet calpain (second-order rate constant = 5.85 mM-1 s-1). P1 also selectively blocked thrombin-induced platelet aggregation. We have now synthesized twenty-three other peptides, analogous to P1, and evaluated them to define the specificity of the amino acid sequence in P1 to selectively block thrombin-induced platelet aggregation. We find that replacement by Leu of Val and by Tyr of Phe adjacent to Gln is minimally tolerated and the resulting peptides are partially effective in selectively blocking thrombin-induced platelet aggregation. The presence of valine adjacent to cysteine in P1 is essential for the inhibitor to selectively block thrombin-induced platelet aggregation. The presence of valine adjacent to cysteine in P1 is essential for the inhibitor to selectively block thrombin-induced platelet aggregation. Extensions of the N-terminal sequence in P1 did not improve its selectivity. Ac-Ala-Gln-Val-Val-Ala-Gly-NH2 (Ac, acetyl), a peptide containing the conserved sequence but lacking the Npys function, neither inhibited platelet calpain nor platelet aggregation induced by thrombin. Presence of the peptide sequence and Npys function are both required in P1 for its selective action in inhibiting platelet aggregation induced by thrombin.