Preparation and partial characterization of two forms of bovine thrombin

Lundblad, Roger L.; Uhteg, Lawrence C.; Vogel, Christine N.; Kingdon, Henry S.; Mann, Kenneth G.

doi:10.1016/0006-291x(75)90536-7

Cited by 187 publications

(48 citation statements)

References 15 publications

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“…Parke-Davis thrombin was further purified by ion-exchange chromatography according to Lundblad et al (11). Thrombin activity was expressed in fibrinogen clotting units (11 Platelets loaded with Indo-1 were diluted to 10 x lo6 per ml in HEPES buffer, pH 7.4. Fluorescence measurements were obtained by using a FACS 440 dual laser system (Becton Dickinson) equipped with a Consort 40/ PDP-11/23 microcomputer (Digital Corp.).…”

Section: Methodsmentioning

confidence: 99%

Flow cytometric measurements of cytoplasmic calcium changes in human platelets

et al. 1988

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Thrombin-induced stimulation of human platelets is accompanied by a dramatic increase in cytoplasmic calcium concentrations followed by a slow decrease. These changes are very rapid, are maximal by 10-15 s, and can be detected with probes such as Indo-1. Suspension studies using spectrofluorometry, which reflect a value which is the average of 3 x lo7 cells per ml, indicate a thrombin dosedependent increase in cytoplasmic calcium at doses up to 0.025 units per ml. We show here, using flow cytometry, that at less than halfsaturating thrombin doses only subpopulations of platelets rather than the entire sample are responding. The extent of these responses, however, still depends on thrombin concentration. When the thrombin doses are between half and fully saturating, one subpopulation responds fully (i.e., its extent of increase in cytoplasmic calcium concentration, [Ca++]i, is 100% of that seen at saturating thrombin concentrations) while the remaining platelets respond partially or not at all. There is thus evidence of positive cooperativity leading to disproportionate thrombin receptor occupancy on different subpopulations when platelets are subjected to subsaturating doses of thrombin. The existence of responding subpopulations may explain how the reported multiple stimulations of the same suspension of platelets at low thrombin doses occur.

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Section: Methodsmentioning

confidence: 99%

Flow cytometric measurements of cytoplasmic calcium changes in human platelets

et al. 1988

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“…Purified RW-X was generously provided by Dr. Walter Kisiel (University ofWashington, Seattle) (14). Bovine thrombin was prepared as described by Lundblad et al (15).…”

Section: Methodsmentioning

confidence: 99%

“…After the collection of blood (six parts) into acid-citrate dextrose anticoagulant (one part), all manipulations were performed at 370C. Platelet-rich plasma was obtained by centrifugation of the anticoagulated blood at 190 g for 15 min. Washed platelets were obtained by use of an albumin (0.35%)/ Tyrode's solution containing apyrase throughout and heparin in the initial wash. After the final wash and centrifugation, the platelets were resuspended in Tyrode's wash medium (0.137 M NaCl, 2.7 mM KCL, 12 mM NaHCO3, 0.36 mM NaH2PO4, 1 mM MgCl2, 5mM dextrose) containing 5 mM Hepes and 3 Ag/ml apyrase, pH 7.35.…”

Section: Methodsmentioning

confidence: 99%

Factor V (Quebec): a bleeding diathesis associated with a qualitative platelet Factor V deficiency.

Tracy¹,

Giles²,

Mann³

et al. 1984

J. Clin. Invest.

130

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Abstract. Studies were performed on a FrenchCanadian family afflicted with a bleeding disorder exhibiting an autosomal dominant inheritance pattern and a severe bleeding diathesis after trauma. Clinical laboratory coagulation tests were unimpressive; the only persistent abnormalities include mild thrombocytopenia and moderately reduced Factor V clotting activities. Some individuals had prolonged Stypven times when platelet-rich plasma was used, suggesting that their platelets could not support functional prothrombinase complex assembly. Detailed studies were performed by use of plasma and isolated, washed platelets from a sister and brother. Bioassay data indicate that both individuals had Factor V activities of -40 and 36% of normal, respectively. A comparison of the Factor V radioimmunoassay and bioassay data on the brother's plasma indicated that the circulating amount of Factor V functional activity was low relative to Factor V antigen concentration (-65-75%). In both individuals, the platelet Factor V functional activities were extremely low (2-4%) relative to antigen levels present as determined by radioimmunoassay. These discrepancies between Factor V activities and antigen concentration do not appear to be due to an unstable Factor V molecule or to the presence of a Factor V or Factor Va inhibitor or inactivator. Kinetics of prothrombin activation by use of purified clotting factors indicated that thrombinactivated platelets from both individuals supported prothrombinase complex assembly identical to controls in the presence of added purified Factor Va. Consequently, their bleeding diathesis appears to reflect their platelet, rather than their plasma, Factor V activity. These results suggest that platelet Factor V is an essential component in maintaining stable and prolonged hemostasis after trauma.

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“…VWF/Factor VIII (10 U/ml) was activated by incubation with bovine a-thrombin (0.01 U/ml) in Tris-buffered saline for 3 min and was used immediately (10,24). Bovine a-thrombin (2.5 NIH U/ag), prothrombin (13 U/mg), and Factor VII (7,100 U/mg) were purified to homogeneity as described previously (25)(26)(27). Thrombin was iodinated by the lactoperoxidase technique (see above); the radiolabeled protein had a specific radioactivity of 4-8 X 103 cpm/ng and the coagulant activity was unaffected by radiolabeling.…”

Section: Methodsmentioning

confidence: 99%