Functional properties and skin care effects of sodium trehalose sulfate.

BACKGROUND: It is known that heparinoid, a mucopolysaccharide polysulfate, is effective in improving rough skin and promoting blood circulation as medicines for diseased areas. However, heparinoid has a molecular weight of more than 5000 and cannot penetrate healthy stratum corneum. OBJECTIVE: We tested the efficacy of sulfated oligosaccharides with a molecular weight of less than 2000 on the human skin barrier function and moisturizing function. METHODS: We measured the transepidermal water loss (TEWL) of a three-dimensional human epidermis model cultured for 3 days after topical application of sulfated oligosaccharides, then observed the effects on TEWL suppression. The mRNA levels of proteins involved in intercellular lipid transport and storage in the stratum corneum, and moisture retention were measured using RT-qPCR. RESULTS: An increase in the mRNA levels of the ATP-binding cassette subfamily A member 12 (ABCA12), which transports lipids into stratum granulosum, was confirmed. Increases were also observed in the mRNA levels of filaggrin (FLG), which is involved in the generation of natural moisturizing factors, and of caspase-14, calpain-1 and bleomycin hydrolase, which are involved in the degradation of FLG. Antibody staining confirmed that the application of sodium trehalose sulfate to 3D model skin resulted in more ABCA12, ceramide, transglutaminase1, and FLG than those in controls. In a randomized, placebo-controlled, double-blind study, participants with low stratum corneum water content applied a lotion and emulsion containing sodium trehalose sulfate to their faces for 4 weeks. Sodium trehalose sulfate decreased the TEWL and increased the stratum corneum water content. CONCLUSION: These results suggest that cosmetics containing sodium trehalose sulfate act on the epidermis by increasing barrier factors and moisturizing factors, thereby ameliorating dry skin.

Development of tight junction-strengthening compounds using a high-throughput screening system to evaluate cell surface-localized claudin-1 in keratinocytes.

Tight junctions (TJs) are important factors constituting the physical barriers of the skin, and their suppression has been described in various conditions, such as aged skin and atopic dermatitis lesions. However, the methods for improving skin TJ function remain insufficient. Therefore, to obtain compounds that can improve TJ function, we developed a novel high-throughput screening system termed live-cell immunostaining to evaluate cell surface-localized claudin-1 (CLDN1) with high selectivity using normal human epidermal keratinocytes (NHEKs). Heparinoid and phospho-pyridoxal (p-Pyr), a metabolite of pyridoxine, were identified as hit compounds. In addition, heparinoid was strongly suggested to increase CLDN1 expression by inhibiting epidermal growth factor receptor signaling. By contrast, p-Pyr did not enhance CLDN1 expression, but it accelerated the translocation of CLDN1 to the cell surface. Finally, we confirmed that heparinoid and p-Pyr improved barrier function in NHEKs in a transepithelial electrical resistance assay. In conclusion, heparinoid and p-Pyr could potentially ameliorate skin conditions by improving TJ function.

Pentosan Polysulfate, a Semisynthetic Heparinoid Disease-Modifying Osteoarthritic Drug with Roles in Intervertebral Disc Repair Biology Emulating the Stem Cell Instructive and Tissue Reparative Properties of Heparan Sulfate.

This review highlights the attributes of pentosan polysulfate (PPS) in the promotion of intervertebral disc (IVD) repair processes. PPS has been classified as a disease-modifying osteoarthritic drug (DMOAD) and many studies have demonstrated its positive attributes in the countering of degenerative changes occurring in cartilaginous tissues during the development of osteoarthritis (OA). Degenerative changes in the IVD also involve inflammatory cytokines, degradative proteases, and cell signaling pathways similar to those operative in the development of OA in articular cartilage. PPS acts as a heparan sulfate (HS) mimetic to effect its beneficial effects in cartilage. The IVD contains small cell membrane HS proteoglycans (HSPGs) such as syndecan, and glypican and a large multifunctional HS/chondroitin sulfate (CS) hybrid proteoglycan (HSPG2/perlecan), that have important matrix-stabilizing properties and sequester, control, and present growth factors from the FGF, VEGF, PDGF, and BMP families to cellular receptors to promote cell proliferation, differentiation, and matrix synthesis. HSPG2 also has chondrogenic properties and stimulates the synthesis of extracellular matrix (ECM) components and expansion of cartilaginous rudiments, and has roles in matrix stabilization and repair. Perlecan is a perinuclear and nuclear proteoglycan (PG) in IVD cells with roles in chromatin organization and control of transcription factor activity, immunolocalizes to stem cell niches in cartilage, promotes escape of stem cells from quiescent recycling, differentiation and attainment of pluripotency and migratory properties. These participate in tissue development and morphogenesis, ECM remodeling and repair. PPS also localizes in the nucleus of stromal stem cells, promotes development of chondroprogenitor cell lineages, ECM synthesis and repair and discal repair by resident disc cells. The availability of recombinant perlecan and PPS offers new opportunities in repair biology. These multifunctional agents offer welcome new developments in repair strategies for the IVD.

Interaction of albumins and heparinoids investigated by affinity capillary electrophoresis and free flow electrophoresis.

A fast and precise affinity capillary electrophoresis (ACE) method has been applied to investigate the interactions between two serum albumins (HSA and BSA) and heparinoids. Furthermore, different free flow electrophoresis methods were developed to separate the species which appears owing to interaction of albumins with pentosan polysulfate sodium (PPS) under different experimental conditions. For ACE experiments, the normalized mobility ratios (∆R/Rf ), which provided information about the binding strength and the overall charge of the protein-ligand complex, were used to evaluate the binding affinities. ACE experiments were performed at two different temperatures (23 and 37°C). Both BSA and HSA interact more strongly with PPS than with unfractionated and low molecular weight heparins. For PPS, the interactions can already be observed at low mg/L concentrations (3 mg/L), and saturation is already obtained at approximately 20 mg/L. Unfractionated heparin showed almost no interactions with BSA at 23°C, but weak interactions at 37°C at higher heparin concentrations. The additional signals also appeared at higher concentrations at 37°C. Nevertheless, in most cases the binding data were similar at both temperatures. Furthermore, HSA showed a characteristic splitting in two peaks especially after interacting with PPS, which is probably attributable to the formation of two species or conformational change of HSA after interacting with PPS. The free flow electrophoresis methods have confirmed and completed the ACE experiments.

Sulfated glycosaminoglycans and non-classically secreted proteins, basic FGF and epimorphin, coordinately regulate TGF-ß-induced cell behaviors of human scar dermal fibroblasts.

BACKGROUND: Upon skin injuries, dermal fibroblasts actively produce transforming growth factor-ß (TGF-ß), which leads to the formation of α-smooth muscle actin (αSMA)-positive granulation tissues. The hyperplasia or incomplete regression of these tissues subsequently causes scar formation in the skin, where sulfated glycosaminoglycans (GAGs), side chains of unique proteoglycans, are supposed to play important roles. OBJECTIVE: The aim of this study is to clarify the effects of sulfated GAGs on dermal cell behaviors triggered by the TGF-ß signaling, along with its possible regulators basic fibroblast growth factor (bFGF) and cell surface epimorphin. bFGF and epimorphin might regulate the TGF-ß-induced αSMA expression, they could exert such effects only in specific cellular contexts, given that they lack conventional signal sequences for extracellular localization. METHODS: Human scar-derived dermal fibroblasts (HSFs) were treated with TGF-ß alone, TGF-ß plus bFGF, and TGF-ß plus cell surface expression of epimorphin. The effects of GAGs on the expression of αSMA and the cellular morphology were then investigated. RESULTS: A highly sulfated chondroitin sulfate (CS-E) or its substitute (heparinoid) had marked inhibitory effects on TGF-ß-mediated changes in HSF behaviors. We found that heparinoid can directly associate with TGF-ß, bFGF and epimorphin. We also found that bFGF downregulated αSMA, which was attenuated by heparinoid, whereas epimorphin augmented αSMA expression, which was further amplified by heparinoid. CONCLUSIONS: TGF-ß, bFGF and epimorphin in the extracellular microenvironment cooperatively affect HSF behaviors under the control of a highly sulfated chondroitin sulfate. These results provide important evidence towards understanding the regulation of TGF-ß-induced HSF behaviors.

Endogenous Heparinoids May Cause Bleeding in Mucor Infection and can be Detected by Nonactivated Thromboelastometry and Treated by Recombinant Activated Factor VII: A Case Report.

Mucormycosis is an aggressive fungal infection, which invades endothelial cells of blood vessels. This condition might lead to destruction of endothelium and release of heparin-like substances to the bloodstream and cause life-threatening bleeding, which is not well described in the literature.We present a patient with mucormycosis who experienced life-threatening bleeding, although no standard laboratory test could detect any coagulopathy.The cause of bleeding-coagulopathy was detected only by nonactivated thromboelastometry (NATEM), which revealed the presence of heparin-like substances. After treatment with recombinant activated FVII rotational thromboelastometry, results improved and the patient stopped bleeding. Regular application of the drug was necessary during acute phase of infection to prevent further bleeding.In this case report, we show that NATEM can detect the presence of heparin-like substances in bleeding patient with mucormycosis infection and that recombinant activated FVII can be used to stop and prevent bleeding until infection resolves.

Superimposed coagulopathic conditions in cirrhosis: infection and endogenous heparinoids, renal failure, and endothelial dysfunction.

In this article, the authors discuss three pathophysiologic mechanisms that influence the coagulation system in patients who have liver disease. First, bacterial infections may play an important role in the cause of variceal bleeding in patients who have liver cirrhosis, affecting coagulation through multiple pathways. One of the pathways through which this occurs is dependent on endogenous heparinoids, on which the authors focus in this article. Secondly, the authors discuss renal failure, a condition that is frequently encountered in patients who have liver cirrhosis. Finally, they review dysfunction of the endothelial system. The role of markers of endothelial function in cirrhotic patients, such as von Willebrand factor and endothelin-1, is discussed.

Heparin-like effect in liver disease and liver transplantation.

Liver cirrhosis is characterized by impairment of primary and secondary hemostasis but it is not clear how this impairment is related to the bleeding problems seen in cirrhosis. This delicate hemostatic balance can be perturbed by numerous conditions, such as variceal bleeding, renal failure, or infection/sepsis, which may lead to worsening of coagulation status to date. The role of endogenous heparinoids (glycosaminoglycans) in the coagulopathy of patients who have cirrhosis has been demonstrated by thromboelastography with the addition of heparinase I in patients who have recent variceal bleeding and infection. The heparin-like effect has also been demonstrated to be part of the coagulopathy seen after reperfusion in patients who have cirrhosis and are undergoing liver transplant. Therapeutic implications of these findings are not clear at the moment and the use of drugs able to cleave heparinoids should be explored.

Characterization of anticoagulant heparinoids by immunoprofiling.

Heparinoids are used in the clinic as anticoagulants. A specific pentasaccharide in heparinoids activates antithrombin III, resulting in inactivation of factor Xa and-when additional saccharides are present-inactivation of factor IIa. Structural and functional analysis of the heterogeneous heparinoids generally requires advanced equipment, is time consuming, and needs (extensive) sample preparation. In this study, a novel and fast method for the characterization of heparinoids is introduced based on reactivity with nine unique anti-heparin antibodies. Eight heparinoids were biochemically analyzed by electrophoresis and their reactivity with domain-specific anti-heparin antibodies was established by ELISA. Each heparinoid displayed a distinct immunoprofile matching its structural characteristics. The immunoprofile could also be linked to biological characteristics, such as the anti-Xa/anti-IIa ratio, which was reflected by reactivity of the heparinoids with antibodies HS4C3 (indicative for 3-O-sulfates) and HS4E4 (indicative for domains allowing anti-factor IIa activity). In addition, the immunoprofile could be indicative for heparinoid-induced side-effects, such as heparin-induced thrombocytopenia, as illustrated by reactivity with antibody NS4F5, which defines a very high sulfated domain. In conclusion, immunoprofiling provides a novel, fast, and simple methodology for the characterization of heparinoids, and allows high-throughput screening of (new) heparinoids for defined structural and biological characteristics.

Bacterial infection in cirrhosis impairs coagulation by a heparin effect: a prospective study.

BACKGROUND: Bacterial infections have been postulated as a trigger for variceal bleeding in cirrhotic patients, and impair coagulation evaluated by thrombelastography (TEG). Endogenous heparinoids have been detected after variceal bleeding and during liver transplantation in some cirrhotics using heparinase-modified-TEG. AIM: To assess if bacterial infection is associated with endogenous heparinoids in cirrhotics, thus impairing coagulation. METHODS: Native and heparinase-modified-TEG (cleavage of heparin and heparan-sulphate) was performed in 60 cirrhotics (Grade A, 2; B, 30; C, 28): 30 infected [septicaemia, 6 (culture positive); 6 (culture negative); spontaneous bacterial peritonitis, 10; chest infection, 4; others, 4], 30 not infected, and five infected patients without liver diseases, comparing TEG parameters r, alpha, and ma. Eight cirrhotics were studied before and after infection. The diagnosis of presence and type of infection was based on international standard criteria. RESULTS: A significant heparin effect was found only in infected cirrhotics (28 of 30) with significant changes in r (P=0.0003), alpha (P<0.0001), and ma (P<0.0001), but in none of those not infected. This effect completely reversed in the eight evaluated after resolution of infection. There was no heparin effect in infected non-cirrhotics. CONCLUSIONS: A heparin effect was only found in cirrhotic patients with infection, further confirming that infection significantly modifies coagulation in cirrhotic patients.

Pentalyte does not decrease heparinoid release but does decrease circulating thrombotic mediator activity associated with aortic occlusion-reperfusion in rabbits.

Hemorrhage and thrombosis are associated with major vascular and trauma surgery. Release of heparinoids and thrombotic mediators may contribute to these complications and have been described in rabbits after aortic occlusion-reperfusion. We hypothesized that the resuscitative fluid used could reduce heparinoid and thrombotic mediator release after aortic occlusion-reperfusion in rabbits as assessed by thromboelastographic variables (R, reaction time; alpha, angle; and G, a measure of clot strength). Anesthetized rabbits were administered lactated Ringer's solution (n = 8) or PentaLyte (n = 8) at reperfusion after 30 min of ischemia. Blood was obtained before ischemia and after 30 min of reperfusion for thromboelastography under four conditions: 1) unmodified sample, 2) platelet inhibition, 3) heparinase, and 4) platelet inhibition and heparinase. During reperfusion, unmodified samples demonstrated a significant increase in R and decrease in alpha and G that was not affected by PentaLyte. In the presence of heparinase, no significant fluid-specific thromboelastographic differences were noted. However, thrombotic mediator release (discerned by a decrease in R and an increase in alpha) during reperfusion in samples with platelet inhibition and heparinase was significantly attenuated by PentaLyte. PentaLyte administration does not decrease heparinoid release but does decrease thrombotic mediator release after aortic occlusion-reperfusion.

Action of human group IIa secreted phospholipase A2 on cell membranes. Vesicle but not heparinoid binding determines rate of fatty acid release by exogenously added enzyme.

Human group IIa phospholipase A2 (hIIa-PLA2) is a highly basic protein that is secreted from a number of cells during inflammation and may play a role in arachidonate liberation and in destruction of invading bacteria. It has been proposed that rodent group IIa PLA2 is anchored to cell surfaces via attachment to heparan sulfate proteoglycan and that this interaction facilitates lipolysis. hIIa-PLA2 contains 13 lysines, 2 histidines, and 10 arginines that fall into 10 clusters. A panel of 26 hIIa-PLA2 mutants were prepared in which 1-4 basic residues in each cluster were changed to glutamate or aspartate (charge reversal). A detailed analysis of the affinities of these mutants for anionic vesicles and for heparin and heparan sulfate in vitro and of the specific activities of these proteins for hydrolysis of vesicles in vitro and of living cell membranes reveal the following trends: 1) the affinity of hIIa-PLA2 for heparin and heparan sulfate is modulated not by a highly localized site of basic residues but by diffuse sites that partially overlap with the interfacial binding site. In contrast, only those residues on the interfacial binding site of hIIa-PLA2 are involved in binding to membranes; 2) the relative ability of these mutants to hydrolyze cellular phospholipids when enzymes were added exogenously to CHO-K1, NIH-3T3, and RAW 264.7 cells correlates with their relative in vitro affinity for vesicles and not with their affinity for heparin and heparan sulfate. 3) The rates of exogenous hIIa-PLA2-catalyzed fatty acid release from wild type CHO-K1 cells and two mutant lines, one lacking glycosaminoglycan and one lacking heparan sulfate, were similar. Thus basic residues that modulate interfacial binding are important for plasma membrane fatty acid release by exogenously added hIIa-PLA2. Binding of hIIa-PLA2 to cell surface heparan sulfate does not modulate plasma membrane phospholipid hydrolysis by exogenously added hIIa-PLA2.

Endogenous heparin-like substances significantly impair coagulation in patients undergoing orthotopic liver transplantation.

UNLABELLED: Orthotopic liver transplantation (OLT) is associated with severe bleeding, especially after reperfusion of the grafted liver. Heparin released from the liver graft contributes to postreperfusion coagulopathy. Although patients with liver cirrhosis have increased levels of endogenous heparinoids, the role of these substances during liver transplantation is unclear. Therefore, we performed native and heparinase-modified thrombelastography (TEG) in 72 patients undergoing OLT. TEG was performed at skin incision, 10 min before and 10 min after clamping of the vena cava, 10 min before and 10 min after graft perfusion, and at the end of surgery. Heparinase-modified TEG compared with native TEG demonstrated heparin activity. In contrast to other investigations, we found significant heparin effects before reperfusion, although patients received no exogenous heparin. These heparin effects were greater in patients with cirrhosis compared with patients with cancer as the underlying disease leading to OLT. Administration of coagulation factors is the usual treatment of coagulopathies during OLT. The comparison of native versus heparinase-modified TEG can distinguish between heparin activity or coagulation factor deficiency as a cause of bleeding complications and provides a rational approach to the treatment of bleeding during OLT. IMPLICATIONS: Impaired coagulation function, contributed to by heparin or heparin-like substances, is frequently observed after reperfusion of a transplanted liver. This study demonstrates that a heparinase-modified thrombelastography can identify significant heparin effects in the absence of exogenous heparin administration in patients undergoing liver transplantation.

Structural analysis of a tumor-produced sulfated glycoprotein capable of initiating muscle protein degradation.

A material of Mr 24,000 has been isolated from a cachexia-inducing mouse tumor (MAC16) and shown to initiate protein degradation in isolated gastrocnemius muscle. Biological activity was destroyed by preincubation with peptide N-glycosidase F (PNGase F) and endo-alpha-N-acetylgalactosaminidase (O-glycosidase) but not by neuraminidase or trypsin. Antibody reactivity was destroyed by treatment with periodate, indicating carbohydrate moieties to be the antigenic determinants. Antigenic activity was also reduced by treatment with PNGase F and O-glycosidase and was completely destroyed by treatment with chondroitinase ABC but was unaffected by treatment with either trypsin or chymotrypsin, confirming that the N- and O-linked sulfated oligosaccharide chains were both the antigenic and biological determinants. Biosynthetic labeling of MAC16 cells using a combination of [35S]sulfate and [6-3H]GlcN gave a single component of Mr 24,000 containing both radiolabels. Similar material could not be isolated from a cell line (MAC13) originating from a tumor that does not cause cachexia in vivo. Digestion of 3H/35S material with PNGase F produced two fragments of Mr 14,000 and 10,000 containing both radiolabels, and digestion with O-glycosidase produced three fragments of Mr 14,000, 6,000, and 4, 000, the first two contained both radiolabels and the third contained only 3H. Digestion of the fragment of Mr 14,000 released by PNGase F with O-glycosidase also gave fragments of Mr 6,000 and 4, 000. The products from both digestions were acidic as determined by anion exchange chromatography on DEAE-cellulose. The negative charge on the fragment of Mr 4,000 was removed by treatment with alkaline phosphatase. This suggests that the charge originated from phosphate residues, and this has been confirmed by biosynthetic labeling of MAC16 cells with [32P]orthophosphate, where radiolabel was incorporated into material of Mr 24,000 and into the fragment of Mr 4,000 after treatment with O-glycosidase. To determine the size of the polypeptide core MAC16 cells were biosynthetically labeled with L-[2,5-3H]His which after chemical deglycosylation produced a major component of Mr 4,000. These results suggest a model for the Mr 24, 000 material consisting of a central polypeptide chain of Mr 4,000 and with phosphate residues that may be attached to the polypeptide or a short oligosaccharide chain containing GlcN, one O-linked sulfated oligosaccharide chain containing GlcN, and of Mr 6,000 and one N-linked sulfated oligosaccharide chain of Mr 10,000 also containing GlcN. Neither chain was cleaved into disaccharides with chondroitinase ABC, suggesting that the material is a sulfated glycoprotein.

Anticoagulant mechanisms of Orgaran (Org 10172) and its fraction with high affinity to antithrombin III (Org 10849).

Orgaran (Org 10172), which has antithrombotic activity in man with apparently minimal bleeding side effects, is a mixture of low-molecular-weight heparan, dermatan, and chondroitin sulfates. The degrees to which the minimum concentrations of Orgaran, its fraction with high affinity for antithrombin III (Org 10849; AT III) and unfractionated heparin, which double the activated partial thromboplastin time (APTT) of pooled normal plasma, inhibit intrinsic activation of factor IX, factor X, and prothrombin were compared. Specific ELISAs were used to quantify the activation of each clotting factor. Factor IX activation, which began without a lag phase, preceded factor X and prothrombin activation by approximately 15 and approximately 25 s, respectively. When used at these functionally equivalent concentrations, heparin (2 micrograms/ml plasma), Orgaran (50 micrograms/ml plasma), and Org 10849 (20 micrograms/ml) could delay the onset of factor IX activation. Compared to control plasma, however, only Orgaran reduced the initial rate of factor IX activation. Heparin and Orgaran delayed the onset of factor X activation by 20 and 15 s, respectively, while Org 10849 could not delay the onset of factor X activation. In addition, each anticoagulant delayed the onset of prothrombin activation. Thus, at concentrations which double the APTT of normal plasma, the combined actions of heparan and dermatan sulfate present in Orgaran can apparently suppress factor IX activation more effectively than heparin, and delay the onset of factor X activation nearly as effectively as heparin. The coordinated inhibition of factor IX and factor X activation by Orgaran may contribute to its antithrombotic effectiveness.

Sertoli cells in culture secrete paracrine factor(s) that inhibit peritubular myoid cell proliferation: identification of heparinoids as likely candidates.

Conditioned medium from Sertoli cells, prepared from testes of 20-day-old rats, contains component(s) that inhibit the incorporation of [3H]-thymidine into DNA of peritubular myoid cells (PMC) and inhibit the proliferation of PMC. These components are trypsin-resistant, heat-stable compounds having a molecular weight less than 30,000. The active inhibitory components in Sertoli cell conditioned medium are inactivated by treatment with heparinase, but not by treatment with hyaluronidase or chondroitin sulfate lyases. Addition of heparin or heparan sulfate results in inhibition of DNA synthesis by PMC in a dose-dependent manner, whereas other glycosaminoglycans (GAGs) examined (hyaluronic acid, keratan sulfate, and chondroitin sulfate) have no detectable effects. Heparin and heparan sulfate are unique among GAGs tested in inhibiting the characteristic multilayer growth pattern of PMC following the attainment of confluence in serum-rich medium. On the basis of these and other data presented, it is concluded that heparin and other heparin-like GAGs synthesized by Sertoli cells are implicated in the modulation of growth of PMC in vitro during co-culture. It is postulated that heparin may play a similar role in maintaining the quiescent peritubular myoid cell phenotype in vivo.

[Transcutaneous penetration of a mucopolysaccharide polysulfuric acid ester in the human. A histochemical study]. / Transkutane Penetration eines Mucopolysaccharidpolyschwefelsäureesters beim Menschen. Eine histochemische Studie.

Using a histochemical test method it was investigated whether a mucopolysaccharide polysulfuric acid ester (MPS, Hirudoid) is able to penetrate the human skin. The penetration of MPS into the corium and the subcutis was demonstrated by metachromatic staining of cells which store MPS, whereby cellular elements acquire a colour which is distinct from the staining solution, as soon as they have incorporated MPS.

Endothelial binding sites for heparin. Specificity and role in heparin neutralization.

The specificity of endothelial binding sites for heparin was investigated with heparin fractions and fragments differing in their Mr, charge density and affinity for antithrombin III, as well as with heparinoids and other anionic polyelectrolytes (polystyrene sulphonates). The affinity for endothelial cells was estimated by determining I50 values in competition experiments with 125I-heparin. We found that affinity for endothelial cells increases as a function of Mr and charge density (degree of sulphation). Binding sites are not specific receptors for heparin. Other anionic polyelectrolytes, such as pentosan polysulphates and polystyrene sulphonates, competed with heparin for binding to endothelial cells. Fractions of standard heparin with high affinity for antithrombin III also had greater affinity for endothelium. However, these two properties of heparin (affinity for antithrombin III and affinity for endothelial cells) could be dissociated. Oversulphated heparins and oversulphated low-Mr heparin fragments had lower anticoagulant activity and higher affinity for endothelial cells than did their parent compounds. Synthetic pentasaccharides, bearing the minimal sequence for binding to antithrombin III, did not bind to endothelial cells. Binding to endothelial cells involved partial neutralization of heparin. Bound heparin exhibited only 5% and 7% of antifactor IIa and antifactor Xa specific activity, respectively. In the presence of 200 nM-antithrombin III, and in the absence of free heparin, a limited fraction (approx. 30%) of bound heparin was displaced from endothelial cells during a 1 h incubation period. These data suggested that a fraction of surface-bound heparin could represent a pool of anticoagulant.

Antithrombin BM from human plasma: an antithrombin binding moderately to heparin.

A human antithrombin was purified app. 60 fold from Cohn fraction IV, to give a single band of about 70.000 molecular weight in polyacrylamide gel electrophoresis. Compared to the similar antithrombin III, this glycoprotein binds only moderately to porcine heparin (hence its name Antithrombin BM), thus requiring higher heparin concentration for full thrombin inhibitor function, and lower ionic strength for elution from a heparin sepharose column. In these respects it resembles "heparin cofactor A", which is, however, characterized by a substantially larger molecular weight. From AT III, AT BM further differs in its absolute dependency on the presence of heparin(oids) for antithrombin activity, in its more pronounced inhibitory specificity largely restricted to thrombin, and in the absence of substantial immunological crossreaction with antibody to AT III. Based on comparative measurements of antithrombin activity in the presence of different amounts of heparin, up to 40% of the antithrombin activity present in human blood may be attributed to AT BM. The in vivo role of this new inhibitor remains to be elucidated.