PubMed Health⌕ Search

Biomedical subjects

D M Tollefsen

Publications and source records attributed to D M Tollefsen.

63 records · Page 4Linked to original sources
PubMed1983

Activation of heparin cofactor II by dermatan sulfate.

We have tested the ability of various glycosaminoglycans to increase the rate of inhibition of thrombin by heparin cofactor II (HCII) and by antithrombin III (ATIII) isolated from human plasma. Heparin, dermatan sulfate, and heparan sulfate from bovine liver (in order of decreasing activity) activated HCII. In contrast, only heparin and bovine liver heparan sulfate activated ATIII, whereas dermatan sulfate was inactive at concentrations less than or equal to 1 mg/ml. Heparan sulfate from human aorta, chondroitin 4-sulfate, chondroitin 6-sulfate, keratan sulfate, and hyaluronic acid had little or no activity with either HCII or ATIII. The second order rate constant for the thrombin-HCII reaction reached a maximum value of 6.4 X 10(8) M-1 min-1 in the presence of 250-500 micrograms/ml of dermatan sulfate compared to 3.8 X 10(8) M-1 min-1 in the presence of 40-80 micrograms/ml of heparin. When 125I-thrombin was incubated with plasma in the presence of greater than or equal to 100 micrograms/ml of dermatan sulfate, the protease became complexed exclusively with HCII, suggesting that HCII is the only thrombin inhibitor in human plasma that can be activated by dermatan sulfate.

Animals↗
PubMed1982

Heparin cofactor II. Purification and properties of a heparin-dependent inhibitor of thrombin in human plasma.

We have isolated a previously unrecognized heparin-dependent inhibitor of thrombin from human plasma. The inhibitor, designated heparin cofactor II (HCII), was purified to homogeneity with sulfated-dextran, DEAE-Sepharose, heparin-Sepharose and Sephadex G-150. HCII is a glycoprotein consisting of a single polypeptide chain with a Mr = 65,600 as determined by sedimentation equilibrium analysis. Other physical properties include s20,w = 4.31 S; Stokes radius = 34 A; E280 1% = 11.7; and pI = 4.95 to 5.15. The purified inhibitor is not precipitated by antibodies directed against seven other plasma protease inhibitors, including antithrombin III (ATIII). HCII blocks the proteolytic and amidolytic activities of thrombin by forming a covalent, 1:1 molar complex with the protease. The second-order rate constant for inhibition of thrombin by purified HCII increases from 5.0 X 10(5) M-1 min-1 in the absence of heparin to 4.5 x 10(8) M-1 min-1 at optimal heparin concentrations of 0.8 to 1.0 unit/ml. In comparison with ATIII, HCII is a relatively ineffective inhibitor of coagulation factor Xa.

Amino Acids↗
PubMed1981

Detection of a new heparin-dependent inhibitor of thrombin in human plasma.

We have demonstrated that human plasma contains a heparin-dependent inhibitor of thrombin that is distinguishable from antithrombin III (AT III). When a 1:50 dilution of plasma was incubated with greater than or equal to 0.01 U/ml heparin and 1 U/ml 125I-thrombin, the labeled thrombin B-chains became incorporated into two complexes of Mr-96,000 and Mr-85,000 that were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and beta-mercaptoethanol. Neither complex was detectable at heparin concentrations less than 0.01 U/ml. When a limiting amount of 125I-thrombin was present, the proportion of radioactivity incorporated into each of the two complexes varied with the heparin concentration. Thus, the Mr-85,000 complex predominated at 0.01-5 U/ml heparin, whereas the Mr-96,000 complex predominated at 5-100 U/ml heparin. The Mr-85,000 complex reacted with antibodies to human AT III and comigrated with the purified thrombin-AT III complex. The Mr-96,000 complex did not react with antibodies to AT III or to alpha 1-antitrypsin, and it was detected in normal quantities after incubating 125I-thrombin with plasma immunodepleted of AT III, alpha 2-antiplasmin, alpha 2-macroglobulin, C1 inactivator, alpha 1-antichymotrypsin, or inter-alpha-trypsin inhibitor. The protein that combines with thrombin to form the Mr-96,000 complex was estimated to be present at a minimum concentration of 90 +/- 26 micrograms/ml (mean +/- SD) in identical to any of the known plasma protease inhibitors and that at relatively high heparin concentrations in vitro it reacts with thrombin more rapidly than does AT III.

Antithrombin III↗
PubMed1976

Evidence for a single class of thrombin-binding sites of human platelets.

We previously demonstrated that human platelets bind a small number of thrombin molecules with high affinity and a larger number with relatively lower affinity (Tollefsen, D. M., Feagler, J. R., and Majerus, P. W. (1974), J. Biol. Chem. 249, 2646). In the present report, equilibrium binding studies using [125I]DIP-thrombin (diisopropylphosphoryl-thrombin) over the range of 0.0002-10 U/ml yield a Hill coefficient of 0.775. Measurements of the rate of dissociation of [125I]DIP-thrombin bound to platelets at relatively high affinity (0.04 U/ml added) indicate a much faster dissociation in the presence of 14 U/ml unlabeled DIP-thrombin (T 1/2 = 1.0 min) than in its absence (T 1/2 - 140 min). [125I]DIP-thrombin bound at low affinity (1.0U/ml added) dissociates from platelets with a T 1/2 = 1.7 min in the absence of unlabeled DIP-thrombin. These results suggest a negative cooperative interaction among receptor sites for thrombin; i.e., as thrombin binds to unoccupied sites, high-affinity receptors are apparently converted to low-affinity receptors. In an attempt to detect whether there is heterogeneity of thrombin receptors, [125I]DIP-thrombin was covalently cross-linked to intact platelets using 1 mM glutaraldehyde. A single complex (apparent molecular weight, 200 000) containing [125I]DIP-thrombin was formed throughout a range of thrombin concentrations in which both high- and low-affinity binding was observed. Since incorporation of [125I]DIP-thrombin into this complex did not occur in the absence of platelets and was inhibited by unlabeled thrombin, the complex may represent thrombin cross-linked to its receptor. We conclude that a single class of receptor sites can account for both high- and low-affinity binding of thrombin to platelets, although interaction between nonidentical sites cannot be excluded.

Binding Sites↗
PubMed1976

The binding of human and bovine thrombin to human platelets.

Human thrombin binds to specific receptors on the surface of human platelets in a manner analogous to bovine thrombin. Thus, two classes of binding are observed--high affinity with a dissociation constant (Kdiss) of 0.02 U/ml and low affinity with a Kdiss of 5 U/ml. Bovine and human thrombin bind to the same platelet receptors, although bovine thrombin binds with slightly greater affinity. When the amount of thrombin bound to platelets is related to the extent of 14C-serotonin release, bovine and human thrombin are equally effective. Antibodies to human and bovine thrombin were found to differ markedly in their ability to precipitate thrombin of the two species. Thus, antibovine thrombin precipitated eightfold more bovine thrombin than human thrombin, while antihuman thrombin precipitated tenfold more human thrombin than bovine thrombin. Similar differences were found in the ability of Fab fragments of these antibodies to block the interaction of thrombin of each species with human platelets. The finding that both species of thrombin, despite significant evolutionary differences in primary structure, retain essentially identical binding sites to platelets suggests that this part of the thrombin molecule is physiologically important and supports our hypothesis of a role for thrombin binding to platelets in platelet function and hemostasis.

Animals↗
PubMed1975

Inhibition of human platelet aggregation by monovalent antifibrinogen antibody fragments.

Monovalent goat antibody fragments (Fab) that were monospecific for human fibrinogen were isolated by affinity chromatography on fibrinogen-Sepharose and used as a direct probe for the involvement of fibrinogen in platelet aggregation and the release reaction. The antifibrinogen Fab inhibited aggregation of washed human platelets induced by thrombin (0.1-10 U/ml) by 50-95%, but had no effect on (14-C)-serotinin release and only a slight inhibitory effect on 125-I-thrombin binding to platelets. Inhibition of aggregation was not observed with nonimmune goat Fab or rabbit antihuman albumie bound tightly at saturation to surface fibrinogen molecules. After washing the platelets once to remove unbound Fab, aggregation by subsequently added thrombin was no longer inhibited. The antifibrinogen Fab inhibited the clotting of fibrinogen by thrombin but did not effect the rate of fibrinopeptide A release, indicating that the Fab inhibits clotting by interfering with the polymerization of fibrin monomers. Our experiments suggest that fibrinogen released from platelets is directly involved in thrombin-induced aggregation of washed platelets, perhaps through polymerization of fibrin monomers generated by proteolytic cleavage of released fibrinogen.

Animals↗
PubMed1975

Binding of the products of prothrombin activation to human platelets.

Binding of prothrombin and activation intermediates 1 and 2 to human platelets was tested with 125-I-labeled protein preparations. None of these precursors of thrombin bound to platelets under conditions in which high affinity binding of thrombin was observed,nor did they cause platelet aggregation or serotonin release. The molecular conformation required for binding to platelets as well as for induction of platelet aggregation and release is present, therefore, only after the final step in prothrombin activation.

Binding Sites↗
PubMed1974

Induction of the platelet release reaction by phytohemagglutinin.

We have previously shown that the erythroagglutinating phytohemagglutinin (E-PHA) from Phaseolus vulgaris binds to the surface of intact human platelets and that adenylate cyclase activity in the particulate fraction of E-PHA-treated platelets is lower than in comparable controls. We now find that E-PHA induces release of [(14)C]serotonin from platelets. Release follows binding of E-PHA, and a haptenic inhibitor of E-PHA binding prevents induction of release. E-PHA does not produce platelet lysis and has little effect on [(14)C]serotonin uptake. Platelets possess approximately 300,000 receptor sites of E-PHA per cell, and we estimate that about 15% of these sites must be occupied by E-PHA to initiate the release reaction. Prior incubation of platelets with prostaglandin E(1), theophylline, or dibutyryl cyclic AMP prevents E-PHA-induced release, although these agents have little effect on E-PHA binding to platelets. Thrombin and E-PHA produce different rates and extents of serotonin release. Thrombin (1 U/ml) causes release of 75-85% of platelet [(14)C]-serotonin, with half-maximal release occurring less than 0.5 min after thrombin addition. E-PHA, however, induces release of only 30-60% of platelet serotonin at a 10-fold slower rate. In addition, utilizing electron microscopy, we have observed striking differences in the morphological changes that occur in platelets exposed to E-PHA as compared with thrombin. Thus, the platelet release reaction may be triggered in part by binding of E-PHA to the cell surface, but this reaction only partially resembles that produced by thrombin.

Blood Platelets↗