title: Modulatory effects of Escherichia coli capsular-derived sulfaminoheparosans and heparins on tissue factor-mediated activation of platelets: flow cytometric analysis.
abstract: Sulfaminoheparosans (alternatively known as bioheparins) represent sulfated derivatives obtained from the K5 capsular polysaccharide of Escherichia coli. Previous studies have shown that these agents are structurally comparable to heparins and capable of exerting anticoagulant and antiprotease effects like heparins. Furthermore they are also able to release tissue factor pathway inhibitor (TFPI). Tissue factor (TF) plays a vital role in the pathogenesis of thrombotic and cardiovascular disorders. Anticoagulants such as heparins and bioheparins inhibit this thrombogenic mediator and thereby downregulate the activation of prothrombin and factor X. This study was carried out to determine the effects of several bioheparin fractions and heparins on TF-mediated platelet activation and their direct effect on platelets using human whole blood flow cytometry. Four different sulfaminoheparosan fractions with mean molecular weights of 20, 9, 7, and 6 kDa were tested for their inhibitory effects on platelet activation at two different concentrations (100 and 10 microg/mL). Unfractionated heparin and a low-molecular-weight heparin, tinzaparin, were also tested under the same experimental conditions for comparative modulatory responses. Fresh whole blood from healthy female and male volunteers (n = 5) was mixed with each of these agents and incubated with TF (diluted thromboplastin C) to activate platelets. Platelets were labeled with the antibodies CD61 FITC (GP IIIa) and CD62 PE (P-selectin). The data were analyzed in terms of percent platelet aggregation and platelet P-selectin expression. At 100 microg/mL, all of these agents strongly and significantly inhibited (approximately 40%) the platelet activation induced by TF in comparison to saline control. The inhibitory effects of each of these agents were slightly weaker (approximately 24% inhibition) at 10 microg/mL. The inhibitory effects of these agents on P-selectin expression correspond to their effects on platelet aggregation. At 100 microg/mL all the agents produced greater than 80% inhibition of P-selectin expression whereas at 10 microg/mL, the inhibition is greater than 70% except for bio-20 kDa, which produced less than 50% of inhibition. No molecular weight dependence was observed with bioheparin fractions in terms of inhibitory effects on platelet aggregation or P-selectin expression. None of the bioheparins and heparins exhibited any direct effects on platelets. These observations suggest that both the bioheparins and heparins are capable of inhibiting TF-mediated activation of platelets. Thus the therapeutic effects of bioheparins in the TF-mediated pathogenesis of platelet activation may be similar to those of heparins.
authors.affiliation: Department of Pharmacology, Loyola University Medical Center, Maywood, Illinois 60153, USA.
authors.forename: Jyothi
authors.initials: J
authors.lastname: Maddineni
authors.forename: Walter P
authors.initials: WP
authors.lastname: Jeske
authors.forename: Francis
authors.initials: F
authors.lastname: Baltasar
authors.forename: Umberto
authors.initials: U
authors.lastname: Cornelli
authors.forename: Marco
authors.initials: M
authors.lastname: Manoni
authors.forename: Debra A
authors.initials: DA
authors.lastname: Hoppensteadt
authors.forename: Jawed
authors.initials: J
authors.lastname: Fareed
mesh_terms: D000925:Anticoagulants / Q000494:pharmacology; D016667:Bacterial Capsules; D004926:Escherichia coli / Q000737:chemistry; D005260:Female; D005434:Flow Cytometry; D006493:Heparin / Q000031:analogs & derivatives* / Q000494:pharmacology*; D006801:Humans; D008297:Male; D008970:Molecular Weight; D019007:P-Selectin / Q000032:analysis; D015539:Platelet Activation / Q000187:drug effects*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011135:Polysaccharides, Bacterial / Q000737:chemistry*; D013925:Thromboplastin / Q000494:pharmacology*
chemical_list: D000925:Anticoagulants; D019007:P-Selectin; D010975:Platelet Aggregation Inhibitors; D011135:Polysaccharides, Bacterial; C050673:capsular polysaccharide K5; D006493:Heparin; D013925:Thromboplastin