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Bovine aortic endothelial cells release a heparin-like substance in the presence of 0.4% fetal calf serum. This substance inhibited the growth of smooth muscle cells in vitro by about 70%. Substitution of platelet-poor plasma for serum resulted in minimal liberation of inhibitory activity from the cells unless at least 10-fold higher concentrations of platelet-poor plasma were utilized. This suggested that a platelet product was involved in the release process. Therefore, we examined the ability of the platelet heparitinase described in the preceding communication to release heparin-like species from cultured endothelial cells. Our results show that when endothelial cells were exposed to serum-free medium containing 1 ng/ml of the purified platelet endoglycosidase, at least as much inhibitory activity was released as was obtained with 0.4% serum. Dose response experiments indicated that only 10 pg/ml of the enzyme were necessary to liberate 50% of the inhibitory activity from endothelial cells. The heparin-like nature of the inhibitory substance was demonstrated by its sensitivity to Flavobacterium heparinase. Utilizing appropriate controls, the release of heparin-like material by the endoglycosidase was shown to be enzyme-specific and was not due to artifacts of experimental manipulations. In addition, this enzyme did not convert prereleased material to an active component, but directly liberated the active heparin-like species from endothelial cells. A simple model describing the possible role of heparin-like components and the endoglycosidase in the normal and injured wall is presented.

Cloned mouse mast cells resemble, by ultrastructure, immature mast cells observed in vivo. These mast cell clones can be grown in the absence of any other cells, facilitating direct investigations of their biochemistry and function. We find that cloned mast cells express plasma membrane receptors (Fc epsilon R) that bind mouse IgE with an equilibrium constant (KA) similar to that of normal mouse peritoneal mast cells. In addition, cloned mast cells do not display detectable la antigens and cannot enhance lg secretion when added to lymphocyte cultures or mediate natural killer lysis. In the presence of 1 mM sodium butyrate, cloned mast cells stop dividing and acquire abundant electron-dense cytoplasmic granules similar to those of mature mast cells. Their histamine content increases concomitant with cytoplasmic granule maturation and may exceed that of untreated mast cells by 50-fold. Unlike peritoneal mast cells, cloned mast cells incorporate 35SO4 into chondroitin sulfates rather than heparin. These findings demonstrate that, unlike fully differentiated mouse peritoneal mast cells, cloned immature mouse mast cells contain no heparin and low levels of histamine. In addition, they establish that high-affinity Fc epsilon R are expressed early in mast cell maturation, well before completion of cytoplasmic granule synthesis and mediator storage.

To assess the contribution of mast cells to the maintenance of blood fluidity, the hindlimb vasculature of mast cell-deficient mice (W/Wv) and littermates containing normal levels of mast cells (+/+), were perfused with purified human thrombin and antithrombin. Enzyme-inhibitor complex generation within the vasculature was enhanced to a comparable extent for W/Wv and +/+ mice over the uncatalyzed rate, that level of complex produced within a similar time interval in the absence of heparin. Perfusion of purified Flavobacterium heparinase prior to infusion of the hemostatic components, or perfusion of antithrombin modified at the heparin-binding domain, reduced W/Wv and +/+ hindlimb thrombin-antithrombin complex formation to the uncatalyzed rate. To further define the cellular source of the vascular-associated heparin-like molecules, endothelial cells isolated from epididymal fat pads of W/Wv and +/+ mice were grown in vitro. The acceleration of thrombin-antithrombin interactions in the presence of endothelial cell-derived glycosaminoglycans was similar for W/Wv and +/+ mice, was abolished with purified bacterial heparinase, and was expressed to only a minor extent when utilizing modified antithrombin. The biologically active mucopolysaccharides appear to be present on the cell surface.

Cloned bovine aortic endothelial cells were cultured with [35S]Na2SO4 and proteolyzed extensively with papain. Radiolabeled heparan sulfate was isolated by DEAE-Sephacel chromatography. The mucopolysaccharide was then affinity fractionated into two separate populations utilizing immobilized antithrombin. The heparan sulfate, which bound tightly to the protease inhibitor, represented 0.84% of the mucopolysaccharide mass, accounted for greater than 99% of the initial anticoagulant activity, and exhibited a specific activity of 1.16 USP units/10(6) 35S-cpm. However, the heparan sulfate that interacted minimally with the protease inhibitor constituted greater than 99% of the mucopolysaccharide mass, represented less than 1% of the starting biologic activity, and possessed a specific anticoagulant potency of less than 0.0002 USP unit/10(6) 35S-cpm. An examination of the disaccharide composition of the two populations revealed that the high-affinity heparan sulfate contained a 4-fold or greater amount of GlcA----GlcN-SO3-3-O-SO3 (where GlcA is glucuronic acid), which is a marker for the antithrombin-binding domain of commercial heparin, as compared with the depleted material. Cloned bovine aortic endothelial cells were incubated with [35S]Na2SO4 as well as tritiated amino acids and completely solubilized with 4 M guanidine hydrochloride and detergents. The double-labeled proteoglycans were isolated by DEAE-Sephacel, Sepharose CL-4B, and octyl-Sepharose chromatography. These hydrophobic macromolecules were then affinity fractionated into two separate populations utilizing immobilized antithrombin. The heparan sulfate proteoglycans which bound tightly to the protease inhibitor represented less than 1% of the starting material and exhibited a specific anticoagulant activity as high as 21 USP units/10(6) 35S-cpm, whereas the heparan sulfate proteoglycan that interacted weakly with the protease inhibitor constituted greater than 99% of the starting material and possessed a specific anticoagulant potency as high as 0.02 USP unit/10(6) 35S-cpm. The high-affinity heparan sulfate proteoglycan is responsible for more than 85% of the anticoagulant activity of the cloned bovine aortic endothelial cells. Binding studies conducted with 125I-labeled antithrombin demonstrated that these biologically active proteoglycans are located on the surface of cloned bovine aortic endothelial cells.

Numerous investigators have postulated that a hypercoagulable state exists in humans for a period of time before the development of thrombotic episodes. A clear biochemical definition of the prethrombotic state, however, has proved elusive due in part to the lack of reliable techniques for monitoring pertinent changes in blood coagulability. Based on recent advances in our knowledge of the biochemistry of the coagulation system, a series of highly sensitive and specific immunochemical tools has been developed that can quantitate the activities of various steps of the hemostatic mechanism in vivo at the subnanomolar level. We have established assays for F1+2 and the protein C activation peptide, which measure the cleavage of the prothrombin molecule by factor Xa and the scission of protein C by the thrombin-thrombomodulin complex, respectively. Nossel and coworkers had previously constructed similar assays for fibrinopeptide A (FPA) and fragment B beta 1-42, which monitor the cleavage of fibrinogen by thrombin and the proteolysis of fibrin I by plasmin, respectively. Substantial elevations in the levels of these markers have been found in patients with disseminated intravascular coagulation and many subjects with acute deep venous thrombosis. The F1+2 and FPA assays have been used to demonstrate that significant increments in factor Xa activity but not thrombin activity regularly occur in the blood of nonanticoagulated individuals with congenital deficiencies of antithrombin or protein C. These two disorders are known to be correlated with the subsequent development of thrombosis. Patients with protein C deficiency have also been noted to have significantly reduced plasma levels of protein C activation peptide. By using the immunoassays for FPA and B beta 1-42 in studies of postoperative patients, it has been shown that an imbalance between the procoagulant action of thrombin and the anticoagulant effect of plasmin on fibrin I polymer may induce an acquired thrombotic diathesis. Finally, we have recently demonstrated that prothrombin activation as measured by the F1+2 assay is suppressed by oral anticoagulants in the blood of patients with thrombotic diatheses. These investigations suggest that these assay techniques can be used to improve our understanding of the hypercoagulable state as well as to develop more effective treatment strategies for the prevention of thromboembolic events.

Affinity-fractionated porcine heparin was randomly scissioned by chemical techniques to give hexasaccharides, octasaccharides, decasaccharides, and mucopolysaccharide fragments of approximately 14 residues and approximately 16 residues that were able to complex with the protease inhibitor. Direct measurements of the kinetic behavior of the hexasaccharides, octasaccharides, and decasaccharides showed that these fractions greatly enhanced the rate of Factor Xa inactivation by antithrombin. Indeed, these species exhibited specific molar activities that ranged from 6.9% (hexaccharide) to 60.9% (decasaccharide) of that of the heparin fragment of approximately 16 residues. However, these oligosaccharides exhibited essentially no ability to accelerate thrombin-antithrombin interactions. The avidity of the hexasaccharides, octasaccharides, and decasaccharides for the protease inhibitor increased as a function of size with the respective dissociation constants ranging from 5.5 X 10(-6) M to 2.9 X 10(-7) M. These data suggest that the region of the heparin molecule needed for catalyzing Factor Xa-antithrombin interaction is intimately related to the antithrombin binding domain. The smallest complex carbohydrate fragment that accelerated the inactivation of thrombin by antithrombin had approximately 14 residues. This fraction had an avidity for the protease inhibitor of 2.8 X 10(-7) M and specific molar activities of 140 units per mumol (thrombin neutralization) and 460 units per mumol (factor Xa inactivation). The largest heparin fragment examined contained approximately 16 residues. This fraction had an avidity for antithrombin of 2.4 X 10(-7) M and specific molar activities of 500 units per mumol (thrombin neutralization) and 560 units per mumol (Factor Xa inactivation). Detailed kinetic analyses showed that these two species are able to directly activate antithrombin to the same extent with respect to thrombin inhibition. However, the larger mucopolysaccharide fragment is also capable of approximating free enzyme with protease inhibitor.

Phase I clinical studies of antithrombin III (ATIII) concentrate demonstrated a mean in vivo incremental recovery of functional activity of 1.4 percent per unit/kg administered, an initial 50 percent disappearance time of 22 hours, and a biologic half-life of 3.8 days. Based on these observations, a treatment regimen designed to maintain plasma ATIII levels between 75 and 120 percent of normal has been developed. None of 10 subjects with congenital ATIII deficiency treated prophylactically had evidence of thromboembolism, including four pregnant women at the time of delivery. Five subjects treated for acute thrombosis and/or thromboembolism, four of whom were pregnant, recovered without further thrombotic extension or recurrence. Heparin resistance was reversed in two subjects, both pregnant. Nine subjects with acquired ATIII deficiency also received ATIII treatment for venous or arterial thrombosis or disseminated intravascular coagulation, all with low plasma ATIII levels. Two subjects with disseminated intravascular coagulation demonstrated improvement, one clinically, the other biochemically. All patients with congenital ATIII deficiency survived, but only five of nine with acquired deficiency survived, highlighting the importance in acquired ATIII deficiency of the underlying disease in prognosis. Survival rate was especially poor in subjects with arterial thrombosis in the setting of low plasma ATIII. Administration of ATIII concentrate was well tolerated. None of the subjects who received ATIII concentrate demonstrated evidence of an infectious transmissible agent. These studies demonstrate that it is now feasible to safely replace the deficient protein in congenital ATIII deficiency, either prophylactically or therapeutically.

To determine the relationship between platelet secretion and prothrombin conversion in whole blood, the release of platelet factor 4 and the generation of a X(a)-specific cleavage product of prothrombin, fragment 1 + 2, were measured during the coagulation of whole blood. There was a parallel increase in the concentration of the two proteins. Over the first 5 min of incubation, platelet factor 4 concentration increased 6 ng/ml per min, and after 6-7 min, the rate of release increased to 750 ng/ml per min. Over the initial 5-7 min of incubation, fragment 1 + 2 concentration increased 1.5 pmol/ml per min with a subsequent increase of 45 pmol/ml per min. Incubation with 10 muM prostaglandin E(1) or 15 muM prostaglandin I(2) inhibited secretion of platelet factor 4 and delayed the onset of the rapid phase of fragment 1 + 2 generation by 8 min, while stimulation of platelet secretion with 1 mug/ml collagen suspension enhanced production of fragment 1 + 2. The addition of either 10 muM epinephrine or 100 ng/ml collagen suspension to whole blood did not affect either platelet factor 4 release or fragment 1 + 2 generation, although the combination of 3 muM epinephrine and 100 ng/ml collagen suspension enhanced platelet release and prothrombin cleavage. The relationship between platelet factor 4 release and prothrombin cleavage was also studied in Factor VIII-deficient blood. When 0.001 U/ml factor VIII activity was present, <80 ng/ml platelet factor 4 were released, and no fragment 1 + 2 was generated after 30 min of incubation. The addition of 0.008-0.08 U/ml Factor VIII activity progressively increased platelet factor 4 release and prothrombin cleavage. Platelet factor 4 release was normal at 0.08 U/ml Factor VIII activity, whereas prothrombin cleavage was still delayed. Very little thrombin, the amount generated by the cleavage of 3-5 nM fragment 1 + 2, was needed to induce release of platelet factor 4.

A sensitive radioimmunoassay (RIA) for the fragment that is liberated from factor X when this zymogen is activated by factor VII/VIIa-tissue factor or factor IXa was developed. Antisera were raised in rabbits to a synthetic 15 amino acid peptide containing the COOH-terminal sequence of the activation fragment coupled to bovine serum albumin with glutaraldehyde. The reactivity of the antibody population obtained toward the factor X zymogen was negligible (less than 1/36,000 that of the activation peptide on a molar basis). However, because other plasma constituents contributed to a nonspecific basal signal in the RIA, a procedure by which the peptide could be reproducibly extracted from plasma was developed. The mean level of this species in normal individuals younger than the age of 40 was 66.4 pmol/L, and elevations up to 550 pmol/L were observed in patients with evidence of disseminated intravascular coagulation. The validity of these measurements of factor X activation is supported by the fact that the RIA signal migrates on reverse-phase high pressure liquid chromatography in a manner identical to that of the native peptide and can be quantitatively recovered. The mean concentration of the activation fragment was markedly decreased to 25.7 pmol/L in patients with hereditary factor VII deficiency (P = .0001 v normal controls), whereas the mean level in subjects with factor VIII deficiency was 61.1 pmol/L (P greater than .1 v normal controls). These data indicate that the basal (ie, in the absence of thrombosis or provocative stimuli) levels of FXP under in vivo conditions result mainly from the activity of the extrinsic pathway.

We have fractionated crude porcine heparin to obtain highly active as well as relatively inactive species of molecular weight approximately 7000 with specific anticoagulant activities of 360 and 12 units/mg, respectively. Nitrous acid degradation of both of these polymers yielded a tetrasaccharide fraction, 1beta, that contained equimolar amounts of iduronic and glucuronic acids, possessed an internal N-acetylated glucosamine, and carried anhydromannitol at the reducing end position. The 1beta tetrasaccharide derived from the highly active heparin, 1betaa, was recovered in a yield of 1.1 mol/7000 daltons. Our analyses indicate that at least 95% of the 1betaa is a single structure that consists of the following unique monosaccharide sequence: L-iduronic acid --> N-acetylated D-glucosamine-6-sulfate --> D-glucuronic acid --> N-sulfate D-glucosamine-6-sulfate. The 1beta tetrasaccharide fraction from relatively inactive mucopolysaccharide, 1betai, was recovered in a yield of 0.3 mol/7000 daltons and was a mixture of several components. Only 8.5% of the 1betai tetrasaccharide fraction exhibited the same uronic acid placement and sulfate group position found in 1betaa. Thus, 2.6% of relatively inactive mucopolysaccharide molecules contain the unique tetrasaccharide sequence found within each molecule of highly active heparin. Given the correlation between abundance of this unique 1betaa tetrasaccharide sequence and biologic potency, we suggest that this structure represents the critical site responsible for anticoagulant activity.

Counterflow centrifugal elutriation and Percoll density gradient centrifugation were employed to prepare cell populations from rat bone marrow that were selectively enriched in the cytoplasmically immature megakaryocytes and depleted of the most mature megakaryocytes. The incorporation of [14C]leucine into the platelet-specific alpha-granule protein, platelet factor 4, as well as the incorporation of [35S]sulfate into platelet proteoglycans synthesized by the maturing megakaryocytes were monitored as markers of cytoplasmic maturation. Rat platelet factor 4 was specifically isolated and characterized by its high affinity for heparin-Sepharose and its amino-terminal sequence homology to human and rabbit platelet factor 4. The [35S]sulfate-labeled proteoglycans were primarily composed of chondroitin 4-sulfate glycosaminoglycans and were identified as platelet granule components by their ability to be secreted by megakaryocytes in response to thrombin or A23187. The production of both components was increased as much as 3-fold in a dose-dependent manner by the addition of picomolar concentrations of purified megakaryocyte stimulatory factor, without a concomitant increase in general protein synthesis. The above results suggest that the megakaryocyte stimulatory factor may regulate the synthesis of platelet granule components by megakaryocytes and hence control the rate and/or extent of cytoplasmic maturation during megakaryocyte development.

An endoglycosidase which cleaves heparin and heparan sulfate was isolated from outdated human platelets by freeze-thaw solubilization, heparin-Sepharose chromatography, DEAE-cellulose chromatography, hydroxylapatite chromatography, octyl-agarose chromatography, concanavalin A-Sepharose chromatography, and Sephacryl S-200 gel filtration. The overall extent of purification of the platelet heparitinase is about 240,000-fold and the overall yield of the enzyme is about 5.6% as compared to the initial freeze-thaw solubilization preparation. The final product is physically homogeneous as judged by disc gel electrophoresis at acidic pH as well as gel filtration chromatography and exhibits an apparent molecular weight of approximately 134,000. Furthermore, our results indicate that the above enzyme is present within platelet lysosomes. The biologic potency of the endoglycosidase was examined as a function of pH. The data show that the platelet heparitinase is maximally active from pH 5.5 to pH 7.5. However, the enzyme possesses minimal ability to cleave heparin at pH less than 4.0 or greater than 9.0. The substrate specificity of the platelet endoglycosidase was determined by identifying susceptible linkages within the heparin molecule that can be cleaved by the above component. Our studies indicate that this enzyme is only able to hydrolyze glucuronsylglucosamine linkages. Furthermore, investigation of the structure of the disaccharide which lies on the nonreducing end of the cleaved glucuronic acid residue suggests that N-sulfation of the glucosamine moiety or ester sulfation of the adjacent iduronic acid groups are not essential for bond scission.