A case can be made for the participation of polymorphonu-clears (PMN) in the initiation and propagation of venous thrombosis. In animal models leukocytes adhered to areas of veins that serve as sites for initiation of thrombi in patients. In addition, PMN are found in white layers of thrombin where they may interact with platelets to attract more of each. This would add bulk and promote coagulation so that red layers are formed. Lidocaine and one of its derivatives inhibited leukocyte adhesion to veins in dogs and lidocaine reduced the incidence of deep venous thrombosis (DVT) in patients after hip replacement, suggesting but not proving that inhibition of PMN adhesion might have contributed. A new approach for preventing PMN contribution to DVT is suggested by recent studies wich identified three families of adhesive receptors (integrins, intercellular adhesion molecules and selectins) on endothelium, leukocytes and platelets. Monoclonal antibodies against β2-integrins on leukocytes reduced leukocyte adhesion, emigration and PMN-dependent tissue injury in infection, inflammation and ischemia-reperfusion injury in animals. Selectins bind to specific carbohydrate ligands containing sialylated Lewis X, suggesting that relatively small analogues might inhibit PMN adhesion. Both platelets and PMN adhere to polymerizing fibrin through undefined mechanisms. Inhibition of this process might inhibit the buildup of white layers of thrombi.
Flavoridin and echistatin, isolated from the-venom of Trh,¢resuruxflararlridis and Echix ¢¢zrh~utu$. respectively. I~lonB ¢o tilt disintel;rin family of inttgrin p~ and 0~ inhibitors ol" low molecular w¢isht gGD.¢ontainin8, cy,qeinc.vith poptid=s. Sin= disulfide bonds ar¢ criti~l for expression ot" biological activity, we sought to determine their location in these two proteins. In fluvoridin, direct cvidcn=: for the exist¢n¢¢ o1. linkug~ between Cys'-Cy= =" and betwcgn Cys ° and Cys" was obtained by analysis of proteolyti¢ products, and indirect evidence sullg¢sts links between Cys'-Cys =+ and Cys~>-Cys ~. In e=histatin, links between Cys'-Cys >~ and Cys'~-Cys ~ were identified by dir=t chemical analysis, 2, MATERIALS AND METHODSSynthetic ethistutin was purchased from Uach=m Inc. (USA) and was used without further purification, Natural echistatin and fla. either natural or synthetic, and flavoridin, (2 rag/m| in lO0 mM NH.,HCO;~. pH 8,0) wcr¢ di;~|cd with TPCK.trypsin (Sigma) at an ¢nxyme:substrato ratio of I:50 (w/w) for 4 h at 3"PC, Dil~tion ol'echistatin was stopl~.d with formic acid (final ¢omazntra. tion 30~ (v/v)), and the styptic popfidcs were scparut¢ by rever~e-phar, e HPLC on a Lichrosphcr JOe RP-l~ (S#M particle size) (Merck, Darmstadt) column cquilibrate.d in a mixture of O.Ig'a (v/v) TFA in wuttr (solution A) and in u=tonilrile (solution B) (95~ A/:~% I~) and ¢luted at I mVmin, at ~rst isocratically for 5 rain and then followed by a linear gradient up to 40'~ B in 70 rain, Th= enzyme in the trypti¢ digest o1" flavoridin was h=at-inactivatcd (1,00"C t'or 5 rain), endnproteinase Asp-N (Boehrin~r.Mannhcim, Indianapolis. IN) was ;tddcd up to u final tnxyme:substra|c ratio o1" h25 (w/w) and incubated 6 h at 37'C, Thereafter, the resuhinll pep tides were separated ;is above. Elation conditions were: 5 rain isoeratirally (100% A) followed by a linear gradient up to 30~ B in 90 rain.The isolated peptid¢s wore chaructedzc.d by amino acid analysis (after acid hydrolysis with 6 N HCI foe 24 h at 110"C using a Biotronic (amino acids aualyzcr) and amino-tem~inal sequence analysis (using an Applied Biosystcms. Sun Francisco. CA) gas-phase scquenc=r mod~l 473A, Mass spectra were recorded with a mats spectrometer MAT 900 (Finni~n MAT. Bremen. FRO) equipped with liquid s¢c-oudaw ion ionization system. RESULTS 3,1. Disu~de bridge pattern of fiavorldinProteolytic digestion of flavoridin with trypsin and endoproteinuse Asp.N gave nine lt'actions (Table I), 316 Published by Efscvicr S¢ietw¢ Publldtcrs B. V,
Previous studies indicate that exposure of fibrinogen receptors associated with glycoprotein lIb/llla complex contributes to platelet loss during cardiopulmonary bypass. Recently, we isolated a number of RGD (Arg-Gly-Asp)-containing, low molecular weight, cysteine-rich peptides from viper venoms. These peptides, which we propose to call "disintegrins," block platelet-fibrinogen interaction and platelet aggregation. We compared the effect of RGDS (Arg-Gly-Asp-Ser) and four disintegrins (echistatin, flavoridin, albolabrin, and bitistatin) on platelet behavior in a membrane oxygenator. During simulated extracorporeal circulation for 2 hours, platelet count decreased to about 30% of initial values. Addition of echistatin (60-200 nM), albolabrin (60-200 nM), bitistatin (60 nM), and flavoridin (45 nM) significantly inhibited platelet loss in the circuit. RGDS (33 /lM) did not show any significant inhibitory effect. ADP-induced platelet aggregation was inhibited in samples of platelet-rich plasma taken from the circuits containing disintegrins. However, echistatin appeared to be a more potent inhibitor of platelet aggregation, whereas albolabrin and flavoridin interfered more selectively with platelet loss from the circuit. Echistatin prevented the accumulation of glycoprotein IlIa on the surface of the circuit. Echistatin (60-200 nM), flavoridin (45 nM), bitistatin (60 nM), and albolabrin (200 nM) significantly inhibited the loss of j-thromboglobulin from platelets into circulating plasma. Electron microscopy studies demonstrated shape change but not degranulation in platelets circulating in the presence of 200 nM echistatin. On the other hand, this peptide (up to 1,000 nM) did not prevent loss of a granules and ,3-thromboglobulin from thrombin-stimulated platelets, although it prevented their aggregation. In conclusion, disintegrins protect platelets in the circuit by preventing their adhesion to surfaces and, therefore, preventing fragmentation of adhered platelets under the shear stress of flowing blood. This study indicates that distintegrins may be potential candidates for platelet protection during cardiopulmonary bypass. (Circulation 1990;82:261-273) Cardiopulmonary bypass involves extensive contact between blood and synthetic surfaces of the membrane oxygenator. Bypass produces thrombocytopenia,1"2 platelet fragmen-
Pretreatment of human platelets with the metabolic inhibitors rotenone and 2-deoxyglucose, before French press homogenization, has led to the isolation of dense storage granules in an overall yield of about 20%. The concentrations of serotonin, ATP and ADP were estimated in the dense granules. Serotonin was 40-60-fold enriched in the dense granules compared to the platelet homogenate. Stored ATP and ADP were also 40-fold enriched in the dense granules compared to the estimated storage nucleotide pool in intact platelets. The ATP to ADP ratio in the isolated dense granules was 0.68-0.70, the same as the ratio of the secreted ATP and ADP. In platelets prelabeled with [SH]adenine, the specific radioactivities of the ATP and ADP in the isolated dense granules and of the secreted ATP and ADP were both negligible, whereas the estimated specific radioactivity of the metabolically active ATP and ADP was 2,000 cpm/nmol. These results confirm that the ATP and ADP in the isolated dense granules are the same as the secreted ATP and ADP in terms of metabolic inactivity and their ATP to ADP ratios. KEY WORDS platelets 9 serotonin granules 9 storage ATP and ADP fraetionation subcellular Blood platelets contain secretory granules which disappear when the cells are treated with appropriate stimuli such as ADP or thrombin (for review, see reference 13). At least two types of such granules are known to be present in platelets as shown by the selective release caused by certain of the inducing agents. ADP, for example, induces the release of serotonin, ATP, ADP, and calcium but not of lysosomal enzymes. It is considered that the former compounds are stored together in one type of granule which is different from the granules that contain lysosomal enzymes which can be released by thrombin. Radioautographic techniques have shown that serotonin is stored in the highly electron-dense granules (9), while electron microprobe X-ray spectroscopy revealed the presence of both calcium and phosphorus in them (21,29). Further evidence for the common storage of these compounds in the dense granules is that platelets which lack dense granules contain very much less ATP, ADP, serotonin, and calcium than normal platelets (12,18,19).Dense granules isolated by subceUular fractionation from the platelets of guinea pig, rabbit (6, 7), and human have been shown to contain serotonin and ATP (8) as well as ADP (14). However, generally, the preparation of dense granules from human platelets has been accompanied by such a J. CELL BIOLOGY 9 The Rockefeller University Press 9
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