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

Shuman, Marc A.; Levine, Shirley P.

doi:10.1172/jci109010

Cited by 53 publications

(26 citation statements)

References 21 publications

(22 reference statements)

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“…Prior investigators have measured either the appearance of a thrombin-related neoantigen or the cleavage of fibrinopeptide A by thrombin (10,12,13,15 antithrombin complexes form in plasma, albeit somewhat more slowly than the prediction derived from experiments with the purified proteins (21). The fact that platelets undergo release when only 3-5 nM F1+2 has been cleaved suggests that some thrombin is being formed, since platelets neither bind nor react to any of the prothrombin intermediates (22).…”

Section: Discussionmentioning

confidence: 99%

“…Shuman and Levine (13) used such radioimmunoassay techniques to study the generation of thrombin during coagulation of native whole blood and were able to closely relate thrombin generation and platelet secretion. They concluded that platelet secretion required thrombin, but that inhibition of platelet release did not influence the rate of thrombin generation (13,14). Kaplan and co-workers (10), who added thrombin to washed platelet suspensions containing fibrinogen, or to citrated plasma, noted that the release of fibrinopeptide A, a thrombin-specific cleavage product, preceded platelet release of 8-thromboglobulin or platelet factor 4 (PF-4) and suggested that fibrinogen was the preferred substrate for exogenous thrombin.…”

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confidence: 99%

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Relationship between Platelet Secretion and Prothrombin Cleavage in Native Whole Blood

Rybak¹,

Lau²,

Tomkins³

et al. 1981

J. Clin. Invest.

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A B S T R A C T To determine the relationship between platelet secretion and prothrombin conversion in whole blood, the release ofplatelet factor 4 and the generation of a Xa-specific cleavage product of prothrombin, fragment 1 + 2, were measured during the coagulation of whole blood. There was a parallel increase in the concentration of the two proteins. Over the first 5 min of incubation, platelet factor 4 concentration increased 6 ng/ml per min, and after 6-7 min, the rate of release increased to 750 ng/ml per min. Over the initial 5-7 min ofincubation, fragment 1 + 2 concentration increased 1.5 pmol/ml per min with a subsequent increase of 45 pmol/ml per min. Incubation with 10 ,uM prostaglandin E, or 15 ,uM prostaglandin I2 inhibited secretion of platelet factor 4 and delayed the onset of the rapid phase of fragment 1 + 2 generation by 8 min, while stimulation of platelet secretion with 1 ,tg/ml collagen suspension enhanced production of fragment 1 + 2. The addition of either 10 ,uM epinephrine or 100 ng/ml collagen suspension to whole blood did not affect either platelet factor 4 release or fragment 1 + 2 generation, although the combination of 3 IuM epinephrine and 100 ng/ml collagen suspension enhanced platelet release and prothrombin cleavage.The relationship between platelet factor 4 release and prothrombin cleavage was also studied in Factor VIII-deficient blood. When 0.001 U/ml factor VIII activity was present, <80 ng/ml platelet factor 4 were released, and no fragment 1 + 2 was generated after 30 min of incubation. The addition of 0.008-0.08 U/ml Factor VIII activity progressively increased platelet factor 4 release and prothrombin cleavage. Platelet factor 4 release was normal at 0.08 U/ml Factor VIII activity, whereas prothrombin cleavage was still delayed. Very little thrombin, the amount generated by Dr. Handin is a recipient of a

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Relationship between Platelet Secretion and Prothrombin Cleavage in Native Whole Blood

Rybak¹,

Lau²,

Tomkins³

et al. 1981

J. Clin. Invest.

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“…Because it stimulates platelet secretion at concentrations that are generated physiologically (1-2 nM), it is likely to be important in the initiation ofthe platelet thrombus formed in response to vascular injury (2,3). The mechanism by which thrombin stimulates platelet secretion is unknown.…”

Section: Introductionmentioning

confidence: 99%

Thrombin-Induced Platelet Secretion

Shuman¹,

Botney²,

Fenton³

1979

J. Clin. Invest.

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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.)

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“…Thrombin converts fibrinogen into fibrin which forms part of the blood clot, 1,2) and activates other blood coagulation factors such as V, VIII, XIII, and protein C.3) Moreover, thrombin also activates blood platelets, 4,5) and acts as a mitogen for various cell types. [6][7][8] Thus, thrombin has become a special target in the design and development of antithrombotic agents.…”

mentioning

confidence: 99%

“…3) Moreover, thrombin also activates blood platelets, 4,5) and acts as a mitogen for various cell types. [6][7][8] Thus, thrombin has become a special target in the design and development of antithrombotic agents.…”

mentioning

confidence: 99%

Amidino-Containing Schiff Base Copper(II) and Iron(III) Chelates as a Thrombin Inhibitor

Toyota

Sekizaki

Takahashi

et al. 2005

CHEMICAL & PHARMACEUTICAL BULLETIN

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Thrombin is a trypsin-like serine proteinase that plays a critical role in the blood coagulation cascade. Thrombin converts fibrinogen into fibrin which forms part of the blood clot, 1,2) and activates other blood coagulation factors such as V, VIII, XIII, and protein C.3) Moreover, thrombin also activates blood platelets, 4,5) and acts as a mitogen for various cell types. [6][7][8] Thus, thrombin has become a special target in the design and development of antithrombotic agents.Various naturally occurring thrombin inhibitors have been found: hirudin (from the leech Hirudo medicinalis), 9,10) cyclotheonamide A, B, and nazumamide A (from the marine sponge Theonella sp.), [11][12][13][14] aeruginosin 298-A, aeruginosins 98-A and 98-B (from the blue-green alga Microcystis aeruginosa). [15][16][17] On the other hand, many compounds having benzamidine or arginine moiety as a partial structure have been synthesized and their inhibitory effects on thrombin examined. [18][19][20][21][22][23] Argatroban (MD-805) proposed by Okamoto et al. 18) is one of most potent synthetic thrombin inhibitors (K i ϭ1.9ϫ10 Ϫ8 M) and is clinically used. Complexes of human or bovine a-thrombin with synthetic arginyl peptides have been analyzed by X-ray diffraction studies. [24][25][26][27][28][29][30] The X-ray data suggested that the thrombin active site consists of the specificity (S1) pocket, the hydrophobic proximal pocket (P-pocket), and the hydrophobic distal pocket (D-pocket). The guanidino nitrogen atoms of inhibitors form a salt bridge with Asp189 (numbering of the thrombin amino acid residues is based on the chymotrypsinogen nomenclature introduced by Bode et al.) 24,28) in S1 pocket, and additional favorable interactions between the inhibitor and D-pocket or P-pocket enhance the inhibitory potency.Cationic organic molecules such as benzamidine and phenylguanidine derivatives are potent inhibitors of trypsin and trypsin-like enzymes, [31][32][33] owing to the electrostatic interaction between the cationic group of inhibitor and an anionic residue at S1 site in the trypsin active site. It is known that salicylaldehyde spontaneously forms a very stable Schiff base metal chelate with an a-amino acid and metal ion. Therefore, we designed and synthesized amidino-or guanidino-containing Schiff base metal chelates (Chart 1) as trypsin-like enzyme inhibitors. [34][35][36] The manifold compounds in the series of A and B can be prepared by changing the combination of salicylaldehyde derivatives, various aamino acids, and metal ions, respectively.Recently we proposed a new binding mode between trypsin active site and amidino-containing Schiff base metal chelate by X-ray diffraction study.37) The cationic amidino group of chelates was shown to form a salt bridge with the carboxylate of Asp189 in the S1 pocket of trypsin as expected. In addition, it was evidenced that the metal ion and the phenolic oxygen of the Schiff base contribute additional interactions with His57 and Ser195 of trypsin.In this report, the inhibitory effect of amidin...

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

Cited by 53 publications

References 21 publications

Relationship between Platelet Secretion and Prothrombin Cleavage in Native Whole Blood

Relationship between Platelet Secretion and Prothrombin Cleavage in Native Whole Blood

Thrombin-Induced Platelet Secretion

Amidino-Containing Schiff Base Copper(II) and Iron(III) Chelates as a Thrombin Inhibitor

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