The perturbation of thrombin binding to human platelets by anions.

A B S T R A C T Factor 0, when preincubated with platelet suspensions, at concentrations as low as 1.2 ,sg/ml, inhibited thrombin-induced platelet aggregation. No inhibition of collagen or arachidonic acid-induced platelet aggregation was found. Inhibition occurred, but to a lesser extent, when thrombin and factor 0 were added to platelets at the same time.No inhibition occurred when factor D was added after thrombin. Thrombin was able to overcome inhibition by factor 0 by increasing its concentration. Diisopropylphosphorofluoridate-inactivated factor 0 also inhibited thrombin-induced platelet aggregation so that enzymatic activity of factor 0 was not required for inhibition. Factor o absorbed with hirudin coupled to Sepharose 6B showed no decrease in inhibitory capacity. 125I-Factor D bound to platelets in a manner suggesting an equilibrium reaction similar to thrombin. At low factor D input, binding was linear, whereas at higher input, binding began to approach saturation. Binding of 1251-labeled thrombin to platelets was inhibited by factor D. Analysis of these data show that factor D does not alter the total number of thrombin molecules which bind to the platelet surface at saturation. However, the dissociation constant for thrombin is altered from 2.78 to 6.90 nM in the presence of factor D (20 gg/ml). Factor D is thus a competitivie inhibitor of thrombin binding, although the affiinity of factor D for the platelet thrombin receptor is much less than that of thrombin. These phenomena occur at physiologic concentrations of factor 0. Therefore, factor D may function in vivo as an inhibitor of platelet aggregation.

Section: Inhibition Of Platelet Aggregation With Factor D)supporting

confidence: 71%

Properdin factor D: effects on thrombin-induced platelet aggregation.

Davis¹,

Kenney²

1979

“…Human alpha and gamma thrombin were prepared as described previously (11,19,20 [1251]thrombin was used the same day of preparation and the DIP-thrombin over a period of 3 wk. Acid soluble collagen was a gift from Mr. Heinz Scheuenstuhl.…”

Section: Methodsmentioning

confidence: 99%

“…Platelets (0.2 ml) were membrane filtered and the retained pH]serotonin was measured in a liquid scintillation counter. Gross counts per minute were corrected for 125I using 1251 standards which were counted by beta and gamma scintillation spectrometry (11).…”

Section: Methodsmentioning

confidence: 99%

“…Thrombin binding is specific, saturable, and reversible (9). The precise correlation that has been demonstrated between the amount of thrombin bound to the platelet and the extent of secretion suggests that binding may be ofphysiological importance (11). However, although it may be the necessary first step in the secretory pathway, binding in itselfis insufficient to induce the release reaction.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Thrombin-Induced Platelet Secretion

Shuman¹,

Botney²,

Fenton³

1979

A B S T R A C T We have studied the interaction between thrombin and washed, human platelets using prostacyclin, a reversible inhibitor ofplatelet secretion. The effect of thrombin is limited to those reactions that are not inhibited by an increased concentration ofplatelet cyclic adenosine 3',5'-monophosphate, because prostacyclin is a potent inducer of the latter. Prostacyclin-treated platelets were briefly (15-30 s) exposed to low concentrations of human thrombin (0.01-0.2 U/ml). After removal of the prostacyclin and thrombin, the platelets were incubated with fresh thrombin. Although they had not undergone the release reaction after the first thrombin incubation, these platelets had a diminished capacity to secrete [3H]serotonin when exposed to thrombin the second time. Refractoriness was concentration dependent: the higher the initial thrombin concentration, the greater the degree of inhibition of serotonin secretion on subsequent thrombin exposure. Inhibition was closely related to the ability of thrombin to induce platelet secretion and not to its esterase or fibrinogen clotting activity. Diisopropyl fluorophosphate-inactive thrombin did not induce refractoriness. Refractoriness to thrombin did not increase when the time of the initial incubation with thrombin was lengthened, nor was it reversible.Inhibition was thrombin specific: serotonin secretion induced by collagen, wheat germ agglutinin, and the ionophore A23187 was minimally affected. For an equivalent amount of thrombin bound, a decrease was observed in serotonin secretion by thrombin-pretreated platelets compared to control platelets. Thus, there is at least one step in the secretory pathway between thrombin binding and regulation of adenylate cyclase. This step appears to transmit the signal that leads to extrusion of intracellular granular contents.A preliminary report of this work was presented at the Annual Meeting of the American Federation for Clinical Research, San Francisco, Calif., 1978, and was published in abstract form in Clin. Res. 26: 357A. (Abstr.)

“…Thrombin binds specifically and reversibly to high affinity receptors on the platelet surface (5)(6)(7)(8). There is good evidence to suggest that binding muist occur before secretion can take place (8,9). Although secretion does not occur if the active site of thrombin is blocked (4), the lack of thrombin turnover anid the requirement for a threshold concentration of thrombin before release can occur (10) suggest that the thrombin-induced release is not a simple enzymatic process.…”

Section: Introductionmentioning

confidence: 99%

Thrombin generation and secretion of platelet Factor 4 during blood clotting.

Shuman¹,

Levine²

1978

Self Cite

A B S T R A C T We have studied the platelet release reaction and thrombin generation during the spontaneous clotting of whole blood in vitro. Both thrombin formation and secretion of platelet Factor 4 were detected at least 12 min before clotting (clotting time, 22-26 min). Initially, at low thrombin concentrations (2-5 ng/ml), there is a small increase in plasma platelet Factor 4 (<1% of the amount present in serum). This is followed by a gradual increase in both platelet Factor 4 and thrombin concentrations over a 12 to 20-min interval. Finally, 5 min before clotting, there is a rapid increase in both thrombin generation and platelet secretion.Thus, we have shown that the release of platelet Factor 4 is a prolonged reaction and the extent to which it occurs parallels thrombin generation. It is only when thrombin concentrations are high (45-90 nglml)-during the period of clot formation-that the major part of platelet Factor 4 secretion occurs. Release of platelet Factor 4, like fibrin formation, occurs in the last step of in vitro coagulation. INTRODUCTIONPlatelets have been assigned a dual role in hemostasis. Initially, after vascular injury, a platelet aggregate or plug is formed on the subendothelial surface. During plug formation, platelets undergo changes that result in the exposure of a phospholipid surface on which Factors X and II are converted to their active forms (1). Several recent observations indicate that the interaction of platelets with clotting factors during thrombin formation is more complex than initially postulated. Osterud et al. (2) have shown that platelets contain an activated form of Factor V and have postulated