Abstracts 919

1lll. H. Fukami, L. Salganicofj and J. Bauer (Specialized Center for Thrombosis Research, Temple University, Philadelphia, PA 19140, U.S.A.): Pig Platelet Amylo-1,6-Glucosidase- Subcellular Localization an1l Possible Activation by Thrombin. (438) Glycogenolysis in washed pig platelets incubated without glucose at 37° for 1 hour was biphasic. The initial rate (4-6 nmoles/min/mg of ) decreased by 70- 80% when approximately 50% of the original glycogen had disappeared. The rate of glycogenolysis in platelets treat,ed with thrombin did not decrease until more than 70% of the glycogen was broken down. These observations suggested that some mechanism to control excessive glycogen depletion exists in platelets and that thrombin can alter this control. Since glycogenolysis requires the action of 2 , phosphorylase (2 .4. 1.1) and amylo-1,6, glucosidase (3 .2.1.33), the debranching , bheir subcellular distribution was inves- tigated. Pig platelets homogenized by a French press were fractionated into a 12,000 g pellet (granules) a 100,000 g pellet (microsomes) and a 100,000 g supernatant fraction, (soluble fraction). The specific activity of phosphorylase in pig platelets was l 00-fold greater than that of amylo-1,6-glucosidase. Less than l% of the phosphorylase was found in the granule fraction, about 3- 4% in the microsomes and 96% in the soluble fraction. Only 2-3% of the amylo-1 ,6-glucosidase was present in the granule fraction, 25-30% in the microsomes, and about 70% in the soluble fraction. Most of the glycogen (70-80%) sedimented at 100,000 g and the rest was solubilized. These findings are compatible with the hypothesis that half of the glycogen in the resting platelet can be readily degraded to a. limit dextrin or inner core by phosphorylaee and part of the amylo-l ,6.glucosidase; the further breakdown of glycogen in thrombin -activated platelets may depend on the exposure of a cryptic porticn of the de branching enzyme, perhaps by the intracellular release of lysosomal enzymes, a large proportion of which is also found in the 100,000 g pellet.

P. N. Walsh and M.S. Lipscomb (Thrombosis Center, Temple Univ., Phila., Pa., U.S.A.): Collagen-Induced Coagulant Activity (CICA) and Factor-V a111l -XI Activities of Platelets all!l Platelet Plasma Membmnes. ( 439) To examine the hypothesis that collagen-stimulated platelets with associated factor XI can initiate intrinsic coagulation by an alternative pathway not requiring factor XII, platelets were washed using an improved method of albumin density gradient separation and assayed for coagulation factors. Factors II, VII, IX and X eluted readily, VIII and XII less readily; whereas V and XI remained associated. Collagen treatment produced a 2-fold increase in platelet-associated V and XI activities and activation of XI. Freezing and thawing platelets increased V activity 3-fold and XI activity 6-fold and these activities sedimented at 100,000 X g. Platelet plasma membranes isolated by sucrose gradient centrifugation after glycerol lysis had PF 3 activity (per mg phospholipid) 5-fold greater than human brain phospholipid (Folch) and contained 0.45 units of V and 0.23 units XI per mg protein. Membranes had no detectable II, VII, VIII, IX, X and XII activities, progressively shortened clotting t.imes when preincubated with XII.deficient plasma, and responded to collagen by enhancing XI activity. Platelets and platelet plasma membranes obtained from a patient with normal hemostasis and without apparant plasma XI had normal CICA and greater than normal XI. These observations support the view that XI is tightly bound to platelet membranes and can be activated by collagen in the absence of detectable XII with resultant initiation of intrinsic coagulation, even in the absence This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. of plasma XI. (Supported by NIH Grant HL 14217 and AHA Grant 73-726.)

J. N. Shanberge and J. Kambayashi (Mount Sinai Medical Center, Milwaukee, ·wisconsin, U.S.A.): Studies on Platelet-Heparin Intemctions Utilizing a Tritium-Labelled Hepa- rin. (440) Intact platelets were separated from platelet-rich plasma by means of sepharose 2B columns according to the method of Tangen, Berman and Marfey. 90% of the platelets 920 Abstracts were recovered, free of plasma . When tho platelet-rich plasma was treated with tritium-labelled h eparin prior to chromatography the platelets obtained by gel filtration contained radioactivity as measured in a liquid scintillation cotmter. Radioactivity also was found in the plasma protein fractions which carne out of the column before the platelet fractions. However, these latter fractions were eluted earlier than active fractions obtained when tritium-labelled heparin alone was chromatographecl. Platelets obtained by gel filtration were treated with tritium-labelled heparin and then rechromatographed. Again platelets with radioactivity were obtained. Treating the platelet-rich plasma with sodium dodecyl sulfate, which alters surface charge, prevents the platelets from taking up heparin while heparin-plasma protein complexing does not seem to be inhibited. Attempts to release heparin from intact platelets has been unsuccessful. W'hether platelets combine with heparin by surface complexing or by absorption has not been determined, but the ability of platelets to take up heparin does not seem dependent on prior heparin-plasma protein complexing.

G. M. W. Cook (Strangeways Research Laboratory, Cambridge, England): and Cell Adhesion. ( 448) Over the last decade the importance of glycoproteins at the cell surface has become increasingly evident. The role of surface heterosaccharides in cell interaction phenomena will be discussed in terms of these macromolecules providing the cell with a recognition surface; the carbohydrate groups of membrane glycoproteins may be expected to provide considerable variation in surface structure, with great economy of means, commensurate with the large number of specific interactions which take place at the cell periphery. Evidence for surface glycoproteins being involved in cell adhesion will be briefly reviewed. That adhesive specificity might be incorporated into the arrangement of sugar residues within the carbohydrate groups of surface heterosaccharides which are recognized by an appropriate glycosyltransferase on the surface of apposing cells, with the formation of mutable adhesions will be detailed by reference to recent experimental evidence, obtained ·with malignant rat dermal fibroblasts. In this type of work the use of exogenous glyco- proteins as model compounds has proved useful, however, the need to isolate endogenous membrane acceptors is evident and the results of current work in this area will be described.

T. Okumum and G. A. Jam·ieson (American Red Cross Blood Research Laboratory, Bethesda, Maryland 20014): Smface Location of the Soluble Glycoprotein of Human Platelets. ( 449) A soluble glycoprotein has been isolated in homogenous fonn from the soluble frsction of human platelet homogenates (FEES Letters 41, 30, 1974). Chicken antiglycoprotein antisera caused the agglutination of intact platelets and showed specificities directed against the glycopeptide and peptide portions of the molecule. Treatment of intact platelets with prior to homogenization results in quantitative removal of from the isolated glycoprotein. Similarly, radioactivity from 14C-glycine ethyl ester is incorporated into the glycoprotein by extracellular labeling with transglutaminase and it is cleaved to the macroglycopeptide by treatment of intact platelets with chymo-

trypsin, which does not cause the release reaction. These data indicate a surface location This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. of the glycoprotein in intact platelets. (Supported, in part, by USPHS grants.)

I. Hagen (Inst. for Thrombosis Res., Rikshospitalet, Oslo 1, Norway): Studies on Some 'fhrombin-Induced Platelet Membrane Alterations. (450) Membrane alterations in connection with thrombin-induced release reaction have been studied with washed, human platelets. The release reaction was induced by 0.01-1.0 NIH units of thrombin, incubated for 3 min in the p resence of 3 mM EDTA. Membrane proteins exposed on the surface were labeled ·with 125I- by the lactoperoxidase-iodination technique.