Protein disulphide isomerase (PDI) activity is released by activated platelets. In this study, PDI was purified from platelets and found to have an apparent mass, pI and N-terminal sequence similar to those for other human PDIs. Rabbit antibodies were generated and used to establish that, on activation, platelets release a protein immunologically identical to PDI in platelets. Approximately 10% of total platelet PDI was released by thrombin and 20% by calcium ionophore. The antibody was used to demonstrate PDI on the external surface of platelets by electron microscopy. Flow cytometry was used to demonstrate that upon activation of platelets with ionophore PDI was released by vesiculation. Since platelets are present and become activated at sites of vascular injury, platelet PDI may play a role in the various haemostatic and tissue remodelling processes in which platelets are involved.
A B S T R A C T The mechanism of stimulus-response coupling in human platelets was investigated with a new instrument that simultaneously monitors aggregation and secretion in the same sample of plateletrich plasma. When platelets were stimulated by high concentrations of ADP, secretion began only after aggregation was almost complete. With lower concentrations of ADP or with epinephrine, biphasic aggregation was observed, and secretion began simultaneously with, or slightly after, the second phase of aggregation. When platelets were stimulated with high concentrations of y-thrombin or A23187, secretion and aggregation began essentially together. With very low concentrations of y-thrombin or A23187, biphasic aggregation was observed with secretion paralleling the second phase. At every concentration of collagen, secretion and aggregation appeared to be parallel events. Under every condition where the beginning of secretion lagged behind aggregation, secretion was dependent upon aggregation and was inhibited by indomethacin; this is referred to as aggregation-mediated platelet activation. When secretion began at the same time as aggregation, it also occurred in the absence of aggregation and was not blocked by indomethacin; this is referred to as directly induced platelet activation. These observations are -consistent with a simple model of platelet stimulusresponse coupling that includes two mechanisms for activation; aggregation-mediated activation is inhibited by indomethacin, while direct activation does not depend upon aggregation and is not inhibited by indomethacin. Secretion and second wave aggregation appear to be parallel events, with little evidence for second wave aggregation being a consequence of secretion as usually described.
The kinetics of the thrombin-induced release of Ca2+ from human blood platelets have been followed using the calcium-complexing dye murexide with a dual-wavelength spectrophotometer. The observed reaction shows three phases, a lag followed by an exponential phase and a final slow zero-order change. From the dependence of the kinetic parameters on concentration, at least four steps must be proposed for the reaction: (i) a rapid formation of a thrombinplatelet complex, (ii) a first-order transformation of the complex, (iii) a slower first-order release of Ca2+, and (iv) slow turnover of thrombin leading to release of additional Ca2+. P JL latelets are anucleate blood cells that play crucial roles in hemostasis and thrombosis by forming compact, adhesive aggregates (for reviews, see Johnson, 1971, Marcus, 1969, and Mustard and Packham, 1970. A variety of physiological agents, such as thrombin, collagen, and ADP, induce the profound morphological and biochemical changes in platelets that lead ultimately to aggregation and contraction of the aggregate. These changes include pseudopod formation, movement of granules, increased metabolism, and the specific release of certain platelet constituents such as adenine nucleotides, calcium, 5-hydroxytryptamine, and certain enzymes (Holmsen et at., 1969). Release of calcium (Murer, 1969) is of particular interest since comparisons with other secretory cells (Douglas, 1968; Stormorken, 1969) and limited experimental evidence (Grette, 1962;Sneddon, 1972) suggest that Ca2+ may be the agent that mediates other changes.The most potent physiological stimulator of platelets is thrombin, the enzyme that catalyzes conversion of fibrinogen to fibrin as well as other reactions in the overall process of blood coagulation. Thrombin is one of a class of proteolytic enzymes that are distinguishable by an active serine believed to function as a nucleophilic catalyst in the hydrolysis of peptides (for review, see Magnussion, 1971). It is similar to trypsin, with specificity for arginyl and lysyl peptide and ester bonds. However, thrombin has a much higher degree of specificity than trypsin. For example, thrombin will hydrolyze only 4 out of 100 or so trypsin-sensitive bonds in fibrinogen. Like other serine proteases, thrombin is inhibited by DFP, a reagent that forms a covalent, inactive diisopropylphosphoryl derivative (DIP-enzyme)* 1 by reacting with the active serine.The mechanism by which thrombin modifies platelets is not known. The fact that the reaction is observed with trypsin but not with DIP-thrombin (Davey and Luscher, 1967
The release of protein disulfide isomerase by activated platelets was hypothesized on the basis of reported intermolecular and intramolecular thiol-disulfide exchange and disulfide reduction involving released thrombospondin in the supernatant solution of activated platelets (Danishefsky, Alexander, Detwiler: Biochemistry, 23:4984, 1984; Speziale, Detwiler: J Biol Chem, 265:17859, 1990; Speziale, Detwiler: Arch Biochem Biophys 286:546, 1991). Protein disulfide isomerase activity, measured by catalysis of the renaturation of ribonuclease inactivated by randomization of disulfide bonds, was detected in the supernatant solution after platelet activation. The activity was inhibited by peptides known to inhibit protein disulfide isomerase; the peptides also inhibited formation of disulfide-linked thrombospondin- thrombin complexes. The reaction catalyzed by the supernatant solution showed a pH dependence distinct from that of the uncatalyzed reaction. The activity was excluded by a 50-Kd dialysis membrane, and it was eluted in the void volume of a gel-filtration column, indicating that it was associated with a macromolecule. The activity was not removed by centrifugation at 100,000 g for 150 minutes indicating that it was not associated with membrane microvesicles. Possible functions for the release of protein disulfide isomerase by activated platelets are discussed.
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