Protein concentration and adsorption time effects on fibrinogen adsorption at heparinized silica interfaces.

Heparin was modified with adipic dihydrazide and covalently linked to surface-activated silica wafers. X-ray photoelectron spectroscopy was used at each stage of derivatization and showed that successful immobilization had taken place. Surfaces were imaged with atomic force microscopy to determine the uniformity of the heparin layer as well as its thickness. In situ ellipsometry was used to estimate layer thickness as well, and to study protein concentration and adsorption time effects on the adsorption and elution kinetics exhibited by human plasma fibrinogen. The adsorbed amount of fibrinogen increased with time and concentration on each type of surface. Under all experimental conditions, fibrinogen adsorbed at a lower rate and to a lower extent on heparinized as compared to unheparinized silica. In addition, buffer elution experiments showed that fibrinogen was less tightly bound to heparinized silica. In order to examine behavior relative to fibrinogen mobility at these interfaces, the sequential adsorption of fibrinogen was recorded. The difference in adsorption rates between the first and second adsorption cycles, evaluated at identical mass density, indicated that post-adsorptive molecular rearrangements had taken place. In general, higher solution concentration and longer adsorption time in the first adsorption step led to more rearrangement, and these history dependent effects were more pronounced on the heparinized silica. These rearrangements are suggested to involve clustering of adsorbed fibrinogen, in this way increasing the amount of unoccupied area at the interface. These rearrangements were presumably facilitated on the heparinized silica by enhanced lateral mobility of fibrinogen at this negatively charged, highly hydrophilic interface.

Incidence of polysaccharide storage myopathy in draft horse-related breeds: a necropsy study of 37 horses and a mule.

Skeletal muscle samples from 38 draft horse-related animals 1-23 years of age were evaluated for evidence of aggregates of glycogen and complex polysaccharide characteristic of equine polysaccharide storage myopathy (EPSSM). Cardiac muscle from 12 of these horses was also examined. Antemortem serum levels of creatine kinase (CK) and aspartate aminotransferase (AST) from 9 horses with EPSSM and 5 horses without EPSSM were compared. Skeletal muscle from 17 horses contained inclusions of periodic acid-Schiff (PAS)-positive, amylase-resistant complex polysaccharide. Similar inclusions were also present in the cardiac muscle of 1 horse. A vacuolar myopathy with aggregates of PAS-positive, amylase-sensitive glycogen was seen in 8 other horses, and these findings are also considered diagnostic for EPSSM. Antemortem serum activities of CK and AST were often higher in EPSSM horses than in horses without EPSSM. Using the presence of amylase-resistant complex polysaccharide as the criterion for diagnosis of EPSSM, the incidence in this population was 45%. Inclusion of horses with aggregates of glycogen but no amylase-resistant complex polysaccharide as representative of the range of pathologic findings in horses with EPSSM resulted in a 66% incidence in this population.

Structural aspects of heparin responsible for interactions with von Willebrand factor.

Unfractionated heparin (UFH) binds von Willebrand factor (vWF) and inhibits the vWF-platelet GP Ib interaction. For vWF, a heparin-binding domain has been identified, but for heparin, the structures that confer such activity are unknown. To investigate this, UFH was depolymerized by methods that yield structurally distinct fragments. The glycosaminoglycans (GAGs) produced were separated into five groups of homogeneous molecular weight (MW). Anti-Xa activity, vWF binding affinity, and vWF-dependent platelet agglutination were measured. Periodate oxidation but not heparinase digestion destroyed anti-Xa activity. At all MWs, periodate conferred greater vWF binding affinity and greater ability to inhibit platelet agglutination than heparinase. As an example, at MW 6100, the binding IC50 was 100+/-19 micromol/L for a periodate-derived GAG and 527+/-70 micromol/L for a heparinase-derived GAG. At the same MW, the agglutination IC50 was 17+/-5 micromol/L for periodate and 135+/-18 micromol/L for heparinase. This suggests that the disaccharide GlcNS[6S]-IdoA2S, destroyed by heparinase but not periodate, is crucial to heparin-vWF interactions. An MW dependency was also noted, with a minimum dodecasaccharide required for activity inhibition. To further investigate the heparin/vWF interaction, affinity fractionation of heparins was performed with an immobilized peptide derived from a heparin-binding domain of vWF. Disaccharide analysis of high-affinity heparins revealed an increased ratio of IdoA2S-GlcN[S/Ac]6S to IdoA2S-GlcN[S/Ac]. Affinity fractionation of oligosaccharides (MW 3500) diminished the relative content of all disaccharides except IdoA2S-GlcNS6S, which was increased. These data suggest that the disaccharide structures IdoA2S-GlcNS6S and GlcNS6S-IdoA2S are crucial to heparin/vWF interactions. Understanding the structural aspects that confer such activity may be useful in designing heparin-based antithrombotic drugs.

Heparins designed to specifically inhibit platelet interactions with von Willebrand factor.

BACKGROUND: Platelet interactions with the injured vessel wall may contribute significantly to the early and late failures of many cardiovascular interventions; the adhesive protein von Willebrand factor (vWF) is thought to play an important role. Previously, we demonstrated that heparin interfered with platelet/vWF hemostatic mechanisms by binding to vWF within the proteins's domain responsible for binding the platelet vWF receptor, glycoprotein Ib. The purpose of the present study was to develop and refine heparins with greater potency to inhibit platelet/vWF interactions. METHODS AND RESULTS: Immobilized synthetic peptides based on a known heparin-binding domain of vWF were used to yield novel fractions of standard heparin that demonstrated a sevenfold increase in their ability to inhibit vWF-dependent platelet agglutination and vWF/platelet binding. The high vWF affinity heparin showed enhanced anti-factor Xa activity but comparable activated partial thromboplastin time activity. Chemical modification of a standard heparin by periodate oxidation and borohydride reduction enhanced its ability to inhibit platelet/vWF interactions by threefold, while eliminating more than 90% of its activated partial thromboplastin time and anti-factor Xa activity. Affinity chromatography of the chemically modified heparin yielded a heparin with an eightfold higher inhibitory potency than the original heparin. CONCLUSIONS: Subspecies of heparin can be developed with significantly enhanced potency to inhibit vWF/platelet interactions. The vWF-inhibiting property of heparin can be dissociated from its antithrombin-binding activity. Based on a growing understanding of heparin/vWF interactions, combinations of affinity separations and chemical modifications could be designed to yield heparins uniquely suitable for prevention of arterial thrombosis.