title: Thrombin facilitates primary platelet adhesion onto vascular surfaces in the absence of plasma adhesive proteins: studies under flow conditions.
abstract: OBJECTIVE
  The effect of local and circulating thrombin on platelet adhesion onto vascular surfaces was explored in the absence of plasma adhesive proteins using flow conditions.
  
  
  METHODS
  To study the local effects of thrombin, denuded rabbit aorta segments were incubated with thrombin concentrations of 0.001, 0.01 and 0.1 U/mL. To evaluate the effects of circulating thrombin, the same concentrations were added to perfusates consisting of washed platelets and washed red blood cells suspended in a human albumin solution (5%). In some experiments, purified von Willebrand's factor (vWF) (Haemate-P) was added to the perfusates (0. 8 U/mL of vWF, final concentration). A humanized chimeric antibody to the GPIIb-IIIa complex (Reopro) was used to determine the role of this glycoprotein on platelet adhesion under the conditions described. The effect of blocking GPIb was also assessed. Perfusions were carried out at 800 s(-1) for 10 min. The interaction of platelet with the vessel surface was morphometrically evaluated and expressed as percentage of surface coverage (%SC). Changes in the surface expression of the major platelet antigens were also analyzed by flow cytometry.
  
  
  RESULTS
  Incubation of subendothelial surfaces with thrombin enhanced platelet deposition with respect to control levels (increases in SC of 64%, 79% and 86% with 0.001, 0.01 and 0.1 U/mL of thrombin, respectively). Low concentrations of thrombin (0.001 and 0.01 U/mL) incorporated in the perfusates resulted in a similar pro-adhesive effect (increases in SC of 64% and 71%, respectively) while the highest concentration (0.1 U/mL) failed to produce a pro-adhesive effect due to the augmented formation of platelet aggregates with subsequent thrombocytopenia (15+/-1 vs. 160+/-5x10(9) plt/L in the perfusates). Similar results were obtained when VWF was present in the perfusate. Reduction of platelet deposition by blockade of GPIIb-IIIa (to 5.3+/-0.7%) was partially restored by thrombin. Blockade of GPIb prevented platelets from adhering even when thrombin was present (%SC of 2.0+/-0.8%). No significant changes in the distribution of platelet membrane glycoproteins during perfusion experiments were detected.
  
  
  CONCLUSIONS
  Our results suggest that thrombin facilitates primary platelet adhesion onto vascular surfaces even in the absence of plasma adhesive proteins. This effect seems to be mainly dependent on the GPIb/vWF axis.
authors.affiliation: Servicio de Hemoterapía y Hemostasia, Hospital Clinic, Universidad de Barcelona, Villarroel 170, 08036 Barcelona, Spain. mdiaz@clinic.ub.es
authors.forename: M
authors.initials: M
authors.lastname: Díaz-Ricart
authors.forename: E
authors.initials: E
authors.lastname: Estebanell
authors.forename: M
authors.initials: M
authors.lastname: Lozano
authors.forename: J
authors.initials: J
authors.lastname: Aznar-Salatti
authors.forename: J G
authors.initials: JG
authors.lastname: White
authors.forename: A
authors.initials: A
authors.lastname: Ordinas
authors.forename: G
authors.initials: G
authors.lastname: Escolar
mesh_terms: D000818:Animals; D016824:Antigens, Human Platelet / Q000378:metabolism / Q000494:pharmacology; D001011:Aorta; D001681:Biological Assay; D015415:Biomarkers / Q000097:blood; D018955:CD36 Antigens / Q000378:metabolism / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000378:metabolism / Q000502:physiology*; D005169:Factor VIII / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D010477:Perfusion; D015539:Platelet Activation / Q000187:drug effects; D010973:Platelet Adhesiveness / Q000187:drug effects*; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism / Q000494:pharmacology; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism / Q000494:pharmacology*; D011817:Rabbits; D013917:Thrombin / Q000494:pharmacology*; D014841:von Willebrand Factor / Q000494:pharmacology
chemical_list: D016824:Antigens, Human Platelet; D015415:Biomarkers; D018955:CD36 Antigens; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D010980:Platelet Membrane Glycoproteins; C083451:human platelet antigen 1b; D014841:von Willebrand Factor; D005169:Factor VIII; D013917:Thrombin