Immunocytochemical localization of fibrinogen, platelet factor 4, and beta thromboglobulin in thin frozen sections of human blood platelets.

Affinity-purified monospecific antibodies against human fibrinogen and the platelet-specific proteins platelet factor 4 and beta thromboglobulin were used to localize these antigens in thin and ultra-thin frozen sections of mildly fixed, washed human blood platelets. By immunofluorescent double-labeling experiments the distribution of fibrinogen was compared to that of platelet factor 4 and beta thromboglobulin. All three antigens occurred in virtually all platelets and showed and identical, dotlike distribution. For immunoelectron microscopy we used protein A-colloidal gold on ultra-thin frozen sections to visualize the specific reaction indirectly. The staining for platelet factor 4, beta thromboglobulin, and fibrinogen localized exclusively over alpha-granules of washed platelets. Within the granules, platelet factor 4 was localized preferentially in the electron dense, alpha-granule nucleoid, whereas fibrinogen was more predominant in the electron-lucent granule periphery. Beta thromboglobulin localization did not show a preferential intragranular distribution.

The isolation and characterisation of a platelet-specific beta-globulin (beta-thromboglobulin) and the detection of antiurokinase and antiplasmin released from thrombin-aggregated washed human platelets.

A protein fraction was isolated from the supernatant of thrombin-aggregated washed human platelets and was shown, by immunodiffusion techniques, to contain a platelet-specific beta-globulin (beta-thromboglobulin) as the major component. A molecular weight of 35 800 was determined for beta-thromboglobin from the measured sedimentation coefficient of3.0 S and Stokes radius of 2.85 nm. Beta-Thromboglobin was detected in the serum from whole blood and the supernatant of 48-h-old platelet-rich plasma and 28-day-old citrated whole blood, but not in platelet-poor plasma. The fraction containing beta-thromboglobulin was shown to possess an antiurokinase activity but was devoid of antiplasmin activity. A further fraction of approximate molecular weight 70 000 was also isolated which contained an antiplasmin but was devoid of antiurokinase activity.

Platelet antiheparin activity. The isolation and characterisation of platelet factor 4 released from thrombin-aggregated washed human platelets and its dissociation into subunits and the isolation of membrane-bound antiheparin activity.

Platelet factor 4 was isolated by gel filtration from the soluble release products of thrombin-aggregated washed human platelets as a proteoglycan-platelet factor 4 complex of molecular weight 358 000, Stokes radius (r-s) of 14.0 nm, sedimentation coefficient (s) of 7.1 S and frictional ratio (f/f-o) of 3.04. The complex was dissociated at high ionic strength (I equals 0.75) and the proteoglycan separated from platelet factor 4 by gel filtration. Platelet factor 4 had a molecular weight of 27 100, r-s of 2.52 nm, s of 2.4 S and f/f-o of 1.26, was insoluble under physiological conditions but readily soluble at pH 3. Under these conditions platelet factor 4 dissociated into four subunits with a molecular weight of 6900, r-s of 1.92 nm, s of 0.8 S, and f/f-o of 1.52. Qualitative N-terminal amino acid analysis showed the presence of glutamic acid or glutamine as the major end group. Platelet factor 4 was compared with protamine sulphate, which has similar biological properties, by electrophoresis at pH 2.2, in which both migrated as single bands but with differing mobility, and by amino acid analysis which showed a more normal distribution of residues than occurred in protamine sulphate. Of the basic amino acids platelet factor 4 (molecular weight 27 100) contained 5.97% arginine, 3.18% histidine, and 12.31% lysine compared to protamine sulphate with 64.2% arginine, 0.6% lysine and no histidine. A partial specific volume (v) of 0.747 was calculated for platelet factor 4 from its amino acid analysis. A membrane fraction with antiheparin activity, an isopycnic density of 1.090-1.110 and r-s of 15-35 nm, was also isolated by sucrose density gradient centrifugation from the ultrasonicated insoluble platelet residue remaining after thrombin-induced aggregation of washed human platelets. Trypsin treatment of the membrane fraction neither solubilised nor destroyed the activity.

The transfer of platelet factor 4 from its proteoglycan carrier to natural and synthetic polymers.

1. Transfer of dansyl-platelet factor 4 complexed with a series of glycosaminoglycans to heparin has been detected and studied by measuring changes in the anisotropy of the dansyl fluorescence. The protein was most easily transferred from chondroitin sulphate and least easily from heparan sulphate. 2. The transfer of the dye-labelled protein from its biological chondroitin 4-sulphate proteoglycan carrier to natural and synthetic anionic polymers was similarily followed. The transfer to heparin and dermatan sulphate was shown to be the same whether 3 mM Ca2+ or 8 mM EGTA was present in the solution. 3. The shapes of the binding curves of the dansyl-factor to the polymers have been compared at I = 0.4M. 4. The observed changes in anisotropy of dye fluorescence have been correlated with the charge density and the stereochemistry of the charged groups of the natural polymers. Large complexes are observed with polymers of high negative charge/weight ratios. Less charged polymers containing disaccharide units of iduronic acid and glucosamine N-sulphate will also from large complexes at I = 0.15 M. 5. It is demonstrated that the release of a platelet factor 4 proteoglycan complex in vivo would result in the transfer of the protein to heparin, moderate quantities of either dermatan or heparan sulphates would not prevent this transfer.

The interaction of platelet factor 4 with heparins of different chain length.

The interaction of platelet factor 4 with heparin of varying chain lengths has been investigated by labelling the heparin with fluorescein isothiocyanate and monitoring the change in anisotropy of fluorescence when the protein is added to a solution of the polysaccharide. The shape of the titration curve depends on the Mr of the heparin and chains of Mr greater than 10 000 showed a definite break when the concentration of polysaccharide and protein became equimolar. Evidence is presented to show that most of the fluorescein label is linked to residual serines on the heparin. Similar break-points were observed if total fluorescence or light-scattering was used to monitor the interaction. Unlabelled heparin was used for the latter method. These results together with those obtained in buffer of high ionic strength lead us to propose a model where the heparin is wrapped around the tetrameric protein.

Low-resolution structure of the complex of human blood platelet factor 4 with heparin determined by small-angle neutron scattering.

Small-angle neutron scattering was used to confirm that human platelet factor 4 was a compact tetrameric globular protein of radius of gyration 1.74 nm and indistinguishable from a sphere. The same technique, when applied to the 1:1 mol/mol complex of platelet factor and heparin of Mr 14000, revealed that the radius of gyration of the particle varied, depending on the relative proportion of 2H2O to H2O in the solvent. Analysis of this variation by the method of Ibel and Stuhrmann (Ibel, K. and Stuhrmann, H.B. (1975) J. Mol. Biol. 93, 255-266) revealed that in the complex the material of greatest neutron-scattering length (the highly sulphated polysaccharide heparin) was furthest from the centre of the particle. This confirms the postulate of Luscombe and Holbrook (Luscombe, M. and Holbrook, J.J. (1983) in Glycoconjugates (Chester, A.M., Heinegård, D., Lundblad, A. and Svensson, S., eds.), pp. 818-819, Secretariat, Lund) that the exact 1:1 mole ratio of heparin (Mr greater than 10 000) to platelet factor in this stable complex arises from the heparin winding around the outside of a globular protein core.

Plasma beta-thromboglobulin in diabetes mellitus.

The plasma beta-thromboglobulin (betaTG) content was measured in 56 diabetic patients with known complications of this disease, including neuropathy, retinopathy, and ischemic skin lesions. Although two patients were found to have elevated levels beyond the normal range, there was no significant difference between the diabetic group as a whole and the group of 35 controls. The significance of these findings with regard to the proposed contribution of small-vessel platelet sequestration in the pathogenesis of late complications of diabetes mellitus is discussed.

Structural and immunological differences between human platelet and endothelial thrombospondins.

The structural and immunological properties of human thrombospondins isolated from platelets and from endothelial cells were compared. Both thrombospondins were digested with either trypsin or thermolysin, in the presence or absence of calcium, then injected onto a Superose 12 gel filtration column. The isolated thermolysin-generated fragments of thrombospondins were identified by radioimmunoassays using either different monoclonal antibodies or a polyclonal antibody directed against platelet thrombospondin. The results show that platelet and endothelial thrombospondins are both partially protected from trypsin digestion in the presence of calcium but have different trypsin and thermolysin fragmentation patterns. The thermolysin-generated fragments from platelet and endothelial thrombospondins are recognized differently by a monoclonal antibody whereas all of them are identified by a polyclonal antibody.

Reductive amination for solid-phase coupling of protein. A practical alternative to cyanogen bromide.

For coupling proteins to Sephacryl gels periodate oxidation of these gels was investigated as an alternative method to cyanogen bromide activation. Optimum conditions were studied with respect to periodate concentration, time of oxidation, pH and type of coupling buffer, concentration of protein, temperature and time of protein uptake, and protein leakage after coupling. The effects of sodium cyanoborohydride and ascorbic acid as reducing agents, and of manganese ions as a potential catalyst were investigated. Using the derived optimum conditions, stable solid-phased antibodies were produced in high yield and used to adsorb factor VIII from plasma. These gels were stable for many weeks, as was the intermediate oxidised gel. Reductive amination for coupling proteins to oxidised Sephacryl gels results in increased binding and lower leakage than is obtained with cyanogen bromide activated agarose.

Human vascular endothelial cells catabolise exogenous glycosaminoglycans by a novel route.

Heparin and other anticoagulant glycosaminoglycans were radiolabelled with 125I and their catabolism by human vascular endothelial cells in culture was studied. Heparin, heparan sulphate and pentosan polysulphate were associated with the cellular fraction and incorporated into the subendothelial matrix, but dermatan sulphate was not found in either fraction. High molecular weight, fully desulphated carbohydrate chains were major catabolic products of all those glycosaminoglycans which were taken up by the cells. Pentosan polysulphate was not degraded further, but the catabolism of heparan sulphate, and to a lesser extent that of heparin, also yielded small oligosaccharides. Thus the first step in catabolism of exogenous glycosaminoglycans by human vascular endothelial cells appears to be complete desulphation, which destroys their biological activity, followed by depolymerisation of the carbohydrate chain. This alternative to the sequential action of lysosomal exoenzymes is dependent upon binding to the cell; thus dermatan sulphate, which is not associated with the cellular fraction, is not catabolised.

Half-life of platelet factor 4 (PF-4) in plasma and platelets from macaca mulatta.

Human platelet factor 4 (PF-4) showed a reaction of complete identity with PF-4 from Macaca mulatta when tested against rabbit anti-human-PF-4. Such immunoglobulin was used for quantitative precipitation of in vivo labelled PF-4 in monkey serum. The results suggest that the active protein had an intra-platelet half-life of about 21 hours. In vitro 125I-labelled human PF-4 was injected intravenously into two monkeys and isolated by immuno-precipitation from platelet-poor plasma and from platelets disrupted after gel-filtration. Plasma PF-4 was found to have a half-life of 7 to 11 hours. Some of the labelled PF-4 was associated with platelets and this fraction had a rapid initial disappearance rate and a subsequent half-life close to that of plasma PF-4. The results are compatible with the hypothesis that granular PF-4 belongs to a separate compartment, whereas membrane-bound PF-4 and plasma PF-4 may interchange.

Interaction of immobilised unfractionated and LMW heparins with proteins in whole human plasma.

The profile of proteins bound to immobilised heparins in hirudin-anticoagulated human plasma was analysed. In molar terms, antithrombin III was the most abundant protein bound to therapeutic doses of unfractionated heparin (M(r) = 12,000), whereas heparin cofactor II constituted < 1% of the protein bound. Histidine-rich glycoprotein was the only plasma protein likely to influence anticoagulant activity by direct competition with antithrombin III, though significant quantities of complement Factor H, fibrinogen, fibronectin, vitronectin and apolipoprotein B were also detected. Only traces of von Willebrand factor, complement factor I, inter-alpha-trypsin inhibitor, alpha 2-macroglobulin, serum amyloid P and transferrin were identified, and neither thrombospondin nor platelet factor 4 were measurable. Binding of both antithrombin III and histidine-rich glycoprotein varied with the ratio of heparin to plasma. Clexane (M(r) = 4,500) also bound antithrombin III, but both histidine-rich glycoprotein and vitronectin were quantitatively significant neutralising proteins. Neutralising proteins dominated the binding profile for Oligo H (M(r) = 2,200).

Spectrophotometric determination of factor Xa generation in factor IX concentrates.

Previous work from this department, concerned with testing the potential thrombogenicity of therapeutic factor IX concentrates, demonstrated that following recalcification of factor IX concentrates thrombin was generated within 3--30 minutes of incubation (Sas el al. 1975). The test developed (known as the TGt 50 test) is a two-stage assay and was thus found to be time consuming, tedious and tended to become inaccurate with long incubation periods and a large number of samples. A semiautomatic version of the test is reported in which the synthetic peptide Bz-ILE-GLU-GLY-ARG-pNA (S-2222) is added to recalcified, diluted factor IX concentrate in the micro-cuvette of a multiple sample recording spectrophotometer. Information can be obtained on (a) the amount of Xa (if any) present prior to recalcification (b) the initial amount of Xa formed and (c) the time taken to activate all factor =X to Xa. Direct graphical interpretation shows a number of qualitative differences between commercial preparations, but by either of the criteria (b) or (c) above, it is possible to place the different products into "activated" and "non activated" groups such that both the Xa generation times and TGt 50 tests identify the same two groups of products. This aggreement also indicates that the TGt 50 test is independent of the intrinsic factor V levels in the various concentrates.

Production of cryoprecipitate of intermediate purity in a closed system thaw-siphon process.

The thaw-siphon procedure for routine production of cryoprecipitate is described briefly, and also its extension to a reproducible procedure for production of cryoprecipitates of intermediate purity. For the latter, the average yield of factor VIII is 60% of that in the prefrozen plasma and approximately 30% is present in the supernatant plasma. The product contains 0.53 units of VIII: C per mg of total protein (of which about 70% is clottable). The mean value of the ratio VIII: C/VIII: RAG is about 0.7. The yield and purity of the product compare favourably with those reported for intermediate purity factor VIII concentrates produced by large-scale plasma fractionation laboratories. The procedure offers an alternative pathway to the production of factor VIII concentrate of intermediate purity and can be operated in any blood bank with a minimum of equipment.

The different forms of antithrombin II in serum.

Antithrombin III (AT III) complexes were isolated from human serum by affinity chromatography and gel filtration. In the first step of the preparation, using heparin-agarose chromatography, we observed that the complexed form of AT III bound less strongly to the gel than the free form and that about half of the AT III was free. With further purification a 2.5 X 10(5) molecular weight complex was isolated. Using 125I labelled human thrombin, this complex was radioactive indicating the presence of thrombin. Only in a synthetic thrombin-AT III system was a 9 X 10(4) molecular weight complex detected, but not in serum. These facts suggest that in serum AT III complexes may exist in a polymeric form. Also, an AT III antigen derived from the original AT III molecule, but not complexed, was isolated which may be a degradation product.

Studies of a fibrinolytic enzyme from the larvae of Lonomia achelous (Cramer) using chromogenic peptide substrates.

A fibrinolytic agent purified from the haemolymph, hair secretion and whole body extract of Lonomia achelous (Cramer) cleaves various chromogenic peptide substrates. The best substrate were found to be pyro-Glu-Gly-Arg-pNA (S-2444) followed by D-Pro-Phe-Arg-pNA (S-2302) and Bz-Ile-Glu-(or) Gly-Arg-pNA (S-2222) designed for urokinase, plasma kallikrein and factor Xa, respectively. Using substrate S-2251 we also found a plasminogen activator.

The absorption, clearance and metabolic fate of dermatan sulphate administered to man--studies using a radioiodinated derivative.

An iodinated derivative of dermatan sulphate was administered by the intravenous, subcutaneous and oral routes to healthy human volunteers in conjunction with unlabelled dermatan sulphate. Following intravenous injection clearance of radiolabel and concentration as measured by competitive binding assay were highly correlated and displayed complex kinetics which were not dose-dependent. Intact 125I-dermatan sulphate was absorbed following both subcutaneous and oral administration, though there appeared to be selective uptake by the gut of a subfraction comprising the smaller or less sulphated molecules. The intact material was subsequently excreted unchanged in the urine. Degradation products of dermatan sulphate were not detected by either gel filtration or affinity chromatography on Polybrene-Sepharose at any time in either plasma or urine, indicating that administered dermatan sulphate is not catabolised by man.

The measurement of heparin and other therapeutic sulphated polysaccharides in plasma, serum and urine.

The competitive binding assay described will specifically and accurately measure concentrations of administered heparin in biological fluids with a sensitivity of 60 ng ml-1. Neither endogenous glycosaminoglycans, nor plasma proteins such as ATIII and PF4 interfere in the assay. Semi-synthetic highly sulphated heparinoids and LMW heparin can also be measured. Using this assay heparin clearance followed simple first-order kinetics over the dose range 100-5,000 units, but the half-life was strongly dose-dependent. There was good correlation with heparin activity measurements by APTT and anti-Xa clotting assays. Plasma concentrations were measurable for at least 5 h following subcutaneous injection of 10,000 units of heparin. Excretion in the urine could be followed after all but the lowest intravenous dose. This assay, used in conjunction with measurements of heparin anticoagulant activity, will be valuable in the elucidation of mechanisms of action of heparin and the heparinoids, and in the assessment and management of problems related to heparin therapy.

In vitro thrombogenicity tests of factor IX concentrates. II: effects of phospholipids and heparin.

Measurement of the total phospholipid (and that portion active in coagulation) in factor IX concentrates revealed no correlation with in vitro tests of potential thrombogenicity, except in the case of the recalcification time and the thrombin generation test which may detect coagulant phospholipid as well as the presence of thrombogenic enzymes. This is probably due to separation of the prothrombin complex proteins from most phospholipid during ion-exchange chromatography. Although low levels of phospholipid remain in the final product these are apparently insufficient to effect appreciable activation of factor IX concentrates despite low levels of antithrombin III. Two tests which measure the formation of thrombin and factor Xa after recalcification of concentrates were affected by the addition of exogenous phospholipid. However this is a relative effect such that differences are quantitative rather than qualitative. Heparin addition during production of factor IX concentrate was found to have only minor effects on the results of in vitro thrombogenicity tests of the final product. This was confirmed in the laboratory by incubation of unheparinised products with heparin for periods of up to 6 hr.

Characterisation of epitopes on human tissue plasminogen activator recognised by a group of monoclonal antibodies.

Seven mouse monoclonal antibodies have been produced against human melanoma tissue plasminogen activator (t-PA). They were specifically bound to 125I t-PA but not 125I urokinase (u-PA) and inhibited t-PA, but not u-PA, activity in plasminogen-rich 125I fibrin wells. Three of the antibodies directly inhibited the amidolytic activity of t-PA and the two most effective also bound near the active site histidine residue as determined by competition experiments using active site blocking agents. Several antibodies interfered with the fibrin binding properties of t-PA. One antibody neither interacted with the active site nor inhibited fibrin binding but still effectively quenched t-PA activity in fibrin wells suggesting that it masks another region of the molecule necessary for effective biological activity.