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Anti-human fibronectin antibodies produced in a goat or in rabbits stimulate the release of serotonin from washed or gelatin/Sepharose-treated human platelets in a dose-dependent manner. This finding led us to propose that fibronectin on the platelet plasma membrane might serve as a collagen receptor on these cells [Bensusan, H. B., Koh, T. L., Henry, K. G., Murray, B. A. & Culp, L. A. (1978) Proc. Natl. Acad. Sci. USA 75, 5864-5868]. To determine whether direct interaction of the antibody with platelet membrane fibronectin was responsible for this stimulation, we prepared proteolytic fragments of the antifibronectin antibodies. Purified F(ab')2 fragments had a greatly diminished ability to elicit degranulation, and rabbit F(ab')2 fragments were totally ineffective in this regard. Preimmune IgG from these sources was capable of inhibiting antibody-induced serotonin release in a dose-dependent manner. Purified antibody Fc fragments also inhibited this stimulation in doses stoichiometrically equivalent to the inhibition seen with preimmune IgG. These results suggest that (i) platelet stimulation elicited by anti-human fibronectin antibody is mediated by the platelet Fc receptor and therefore is likely to be dependent on antigen-antibody complex formation and (ii) fibronectin on the platelet surface may not be the primary receptor for platelet adherence to collagen.

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Protein phosphorylation was investigated in human platelets after stimulation to secretion by thrombin. After stimulation by thrombin at 4 degrees C (in which secretion is inhibited), phosphorylations of the 80, 56, and 38 kDa polypeptides and dephosphorylation of the 67 kDa phosphopeptide eventually occurred. The phosphorylations of the 27 and 20 kDa polypeptides remained inhibited until the temperature was increased to 37 degree C, which also resulted in secretion. Various stimulants and inhibitors of platelet function were used to characterize individual protein phosphorylations. The divalent-cation ionophore, A23187, induced the phosphorylations (or dephosphorylation) of the same proteins as thrombin with the exception of the 80 kDa protein, which remained incompletely phosphorylated. The intracellular calcium antagonist, TMB-8, inhibited thrombin-stimulated secretion and phosphorylation of all the polypeptides except the 80 kDa protein. The dephosphorylation of the 67 kDa phosphoprotein was not affected by TMB-8. Incubation of platelets with prostaglandin E1 and isobutylmethylxanthine inhibited thrombin-stimulated secretion and the phosphorylation of the 38 and 20 kDa protein and increased the phosphorylation of the 67 and 27 kDa phosphoproteins. These observations may be used to correlate protein phosphorylation with secretion, suggesting a possible sequence of intracellular events that mediate thrombin-stimulated secretion.

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Evidence is presented that fibronectin is present on the platelet cell membrane and that it is a receptor for collagen in the platelet-collagen interaction. First, sodium dodecyl sulfate/acrylamide gel electrophoresis was performed on the proteins remaining attached to the surface of collagen after the removal of the remainder of the platelet by sonication. The material was relatively enriched in a glycoprotein that comigrated with cold-insoluble globulin (CIG), a form of fibronectin, and in other proteins which comigrated with myosin, actin, and tropomyosin. The presumptive presence of contractile proteins is consistent with the presence of microfibrillar proteins. Second, the collagen-attached material was shown to contain a protein that reacted with anti-CIG serum by immunoelectrophoresis. Third, when CIG was preincubated with fibrous collagen, the platelet-collagen interaction was inhibited. Fourth, rabbit anti-human CIG stimulated human platelets to secrete the contents of their dense granules. The stimulation was not due to antibody complexes present in the solution. Fifth, a protein was extracted from well-washed platelets and purified on affinity columns of anti-CIG antibodies. The isolated protein was found to bind to fibrous collagen.

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It has been proposed that the platelet : collagen interaction is mediated in part by the collagen carbohydrate residues. To test this hypothesis we have oxidized monomeric and polymeric collagen with sodium periodate under conditions specifically designed to minimize destruction of periodate-susceptible bonds other than in the carbohydrate residues. Oxidation of the collagen significantly reduced its ability to interact with platelets. The extent of inhibition paralleled the extent of carbohydrate destruction. Oxidation with periodate also delayed the polymerization of the monomeric collagen, but even after polymerization the oxidized collagen failed to initiate the release reaction. These observations suggest that the collagen carbohydrate residues may be either near to or part of the site(s) on the collagen molecule required for platelet adhesion.

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A method for studying the platelet:collagen interaction is described that permits simultaneous measurement of platelet adhesion to collagen and the collagen-initiated release reaction. Plasma-free platelets are passed through short columns composed of polymeric collagen covalently linked to agarose (Sepharose 2B). EDTA (0.3 mM) is used to prevent platelet aggregation. 14C-Serotonin is used to measure the extent of the release reaction. Measurement of adhesion is based upon 51Cr. In the experiments that are described, the extents of both adhesion and serotonin release were a function of the total collagen content of the column and of the number of platelets applied. Up to 100 per cent of the applied platelets adhered to the columns. As much as 70 per cent of the platelet serotonin was released. Intracellular 51Cr, lactate dehydrogenase, and pyruvate kinase, on the other hand, were not lost from the platelets. Plasma-free platelets were prepared by two different techniques: gel filtration and differential centrifugation. Both preparations gave the same results. The influence of the column temperature was also examined. At temperatures below 37 degrees C., there was a sharp drop in serotonin release, but only a slight decline in platelet adhesion to collagen. Our results suggest that the collagen/Sepharose assay system should provide a usable and greatly needed technique for studying the molecular basis for the platelet:collagen interaction for assessing platelet function in abnormal states and for investigating the mechanism of action of potential inhibitors.

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By continuous monitoring of viscosity during the sol-gel transformation of deoxygenated sickle hemoglobin a time: viscosity profile has been demonstrated that can be subdivided into: (1) initial lag phase, (2) gradual and minor increase in viscosity, (3) rapid and major (180 times initial value) increase in viscosity, (4) moderately rapid decrease in viscosity, and (5) achievement of equilibrium at approximately 50 per cent of maximum viscosity increase. The duration of the lag phase, rate of increase in viscosity, and maximum change are greatly influenced by hemoglobin concentration and markedly altered by temperature. Admixtures of hemoglobins A and F lengthen the lag phase and attenuate the rate of increase and magnitude of viscosity change according to proportions added and capacity to interact with deoxyhemoglobin S, but the general configuration of the curve is maintained. A different time: viscosity profile is obtained for mixtures of S and C hemoglobin that is lacking the phase with decreasing viscosity. Relevance to the pathophysiology of the sickling phenomenon is evidence because the quantitative and qualitative changes induced by variations in concentration of deoxygenated hemoglobin S, temperature, amount and type of admixed hemoglobin (A, C, and F), ionic strength, and 2, 3-DPG are in agreement with their known effects upon the sickling of intact cells and upon the minimum gelling point of deoxyhemoglobin S. No final conclusions can be drawn concerning the extent or form of hemoglobin aggregation present in the various phases of the time: viscosity profile; however, the technique lends itself readily to obtaining samples at various points along the curve for additional studies such as electron microscopy and light scattering.

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1). During the sol to gel transformation of deoxygenated sickle hemoglobin, a time-dependent process preceding gel formation (lag phase) was demonstrated that was inversely proportional to a function of the hemoglobin concentration and that occurred without alteration in temperature, pH, or oxygen tension. 2). As determined by the Schachman modification of the capillary viscometer, preparations of oxyhemoglobin S and A and deoxyhemoglobin A were indistinguishable when compared over a wide range of concentrations. Up to the concentration at which gelling occurred, deoxyhemoglobin S exhibited the same viscosity behavior. The viscosity of deoxygenated hemoglobin S within the lower gelling concentration range was normal during the lag phase and became abnormally high only at the time of gelation.

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Acetic anhydride in anhydrous acetone-triethylamine reacts with decalcified bone collagen in two ways. It completely acetylates the e-amino groups of lysine and hydroxylysine and causes a progressive change in the protein matrix, such that increasing amounts of degraded peptides become soluble in alkaline phosphate solution. The peptides produced have molecular weights ranging from 100,000 to less than 2,000 daltons. The number of peptides obtained indicates that there are about 10 bonds cleaved per chain. Ball-mill grinding, particle size of the decalcified bone fragments, and age of the chicken do not affect the fraction of collagen that can be solubilized. The appearance of peptides begins at the point where 90% of the e-amino groups are modified. Both the phosphate-soluble and phosphate-insoluble fractions were essentially completely N-acetylated. Calcified bone under the same conditions is rapidly acetylated to only 45% of the total e-amino groups. With further additions of reagent, the degree of modification of the phosphate-insoluble matrix stabilizes at 50 +/- 4%. At the same time, progressively larger amounts of peptides, soluble in alkaline phosphate solution, are produced. The peptides, which approach 100% N-acetylation, have a distribution of molecular weight from 2000 to 75,000 daltons, indicating 10-15 disrupted peptide bonds per chain. The peptides are initially derived from the more accessible regions of the matrix and subsequently are produced from increasingly inaccessible regions. Chromatography of samples of calcified bone collagen, which were acetylated nearly to completion, indicate that the lysyl and hydroxylysyl residues which are most difficult to modify in the calcified bone are located along continuous regions of the collagen chains. Chromatography of partially acetylated samples also show that all regions of the chains are not uniformly accessible to modification, and further, no single region of every alpha chain is exposed to an identical environment.

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WE HAVE INVESTIGATED WHETHER COLLAGEN QUETERNARY STRUCTURE IS REQUIRED FOR THE PLATELET: collagen interaction. Quaternary structure refers to the assembly of collagen monomers (tropocollagen) into polymers (native-type fibrils). Purified monomeric collagen was prepared from acetic acid extracts of fetal calfskin. Polymeric collagen was prepared by dispersion of bovine Achilles tendon collagen and by incubation of monomeric collagen at 37 degrees C and pH 7.4. The state of polymerization was confirmed by electron microscopy. Release of platelet serotonin in the absence of platelet aggregation was used to determine the effectiveness of the platelet: collagen interaction. All forms of collagen produced serotonin release only after a lag period, but polymeric collagen gave a shorter lag period than did monomeric collagen. Monomeric collagen was also quanidinated selectively to convert collagen lysine groups to homoarginine, while leaving the arrangement of polar groups intact. Guanidination of monomeric collagen increased the rate of polymerization and reduced the lag time in serotonin release. Glucosamine (17 mM) retarded polymerization and inhibited the release of platelet serotonin by monomeric collagen but had little effect on release produced by thrombin or polymeric collagen. At the same concentration, glucosamine did not reduce the sensitivity of platelets to stimulation by collagen or block the platelet: collagen interaction. The only effect of glucosamine was on the collagen: collagen interaction. Galactosamine had a similar effect, but glucose, galactose, and N-acetylglycosamine had no effect. We conclude from this data that collagen monomers cannot effectively interact with platelets and that, therefore, collagen quaternary structure has a role in the recognition of collagen by platelets.

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