Download PDF
Thromb Haemost 1993; 70(06): 1024-1029
DOI: 10.1055/s-0038-1649719
Original Article
Fibrinolysis
Schattauer GmbH Stuttgart

Thapsigargin Amplifies the Platelet Procoagulant Response Caused by Thrombin

E F Smeets
1   The Dept. of Biochemistry, Cardiovascular Research Institute Maastricht, University of Limburg, The Netherlands
,
J W M Heemskerk
2   The Dept. of Human Biology, Cardiovascular Research Institute Maastricht, University of Limburg, The Netherlands
,
P Comfurius
1   The Dept. of Biochemistry, Cardiovascular Research Institute Maastricht, University of Limburg, The Netherlands
,
E M Bevers
1   The Dept. of Biochemistry, Cardiovascular Research Institute Maastricht, University of Limburg, The Netherlands
,
R F A Zwaal
1   The Dept. of Biochemistry, Cardiovascular Research Institute Maastricht, University of Limburg, The Netherlands
› Author Affiliations
Further Information

Publication History

Received 18 May 1993

Accepted after revision 19 August 1993

Publication Date:
06 July 2018 (online)

Also available at
  PDF Download  Permissions and Reprints

Summary

The platelet procoagulant response involves an increase in surface-exposed phosphatidylserine, which allows binding and assembly of enzyme complexes of the coagulation pathway resulting in acceleration of the clotting process. This response essentially requires the presence of extracellular Ca2+, and varies in extent with the type of agonist used. In the present paper we demonstrate that the moderate procoagulant response of human platelets caused by thrombin is strongly amplified by the presence of thapsigargin, an inhibitor of the microsomal Ca2+-ATPase. Thapsigargin, like thrombin, has only a weak effect on procoagulant activity. The large increase in procoagulant activity observed with the combined action of these two agonists is associated with increased shedding of microvesicles from the platelet plasma membrane as well as with inhibition of transport of a fluorescent-labeled analog of phosphatidylserine from the outer to the inner leaflet of the plasma membrane by the aminophospholipid translocase. The latter two observations support current concepts regarding the mechanism of development of procoagulant activity.

Although the synergistic effect of thapsigargin on thrombin-induced procoagulant activity is at least in part due to the high levels of intracellular [Ca2+] evoked by these agonists, the data clearly indicate that a rise of the intracellular [Ca2+] is insufficient to completely explain this response. The present findings suggest that additional factors control expression of procoagulant activity upon stimulation of platelets by thrombin.

 

  • References

  • 1 Rosing J, van Rijn JLML, Bevers EM, van DieijenG, Comfurius P, Zwaal RFA. The role of activated human platelets in prothrombin and factor X activation. Blood 1985; 65: 319-322
    PubMedGoogle Scholar
  • 2 Bevers EM, Rosing J, Zwaal RFA. Platelets and coagulation. In: Platelets in Biology and Pathology MacIntyre DE, Gordon JL. (eds) Res Monogr Cell Tissue Physiol. 1987; Vol3, 13: 127-160
    PubMedGoogle Scholar
  • 3 Bevers EM, Comfurius P, Zwaal RFA. Changes in membrane phospholipid distribution during platelet activation. Biochim Biophys Acta 1983; 736: 57-66
    CrossrefPubMedGoogle Scholar
  • 4 Verhallen PFJ, Bevers EM, Comfurius P, Zwaal RFA. Correlation between calpain-mediated cytoskeletal degradation and expression of platelet procoagulant activity. A role for the platelet membrane-skeleton in the regulation of membrane lipid asymmetry?. Biochim Biophys Acta 1987; 903: 206-217
    CrossrefPubMedGoogle Scholar
  • 5 Jackson TR, Patterson SI, Thastrup O, Hanley MR. A novel tumour promotor, thapsigargin, transiently increases cytoplasmic free Ca2+without generation of inositol phosphates in NG115-401L neuronal cells. Biochem J 1988; 253: 81-86
    CrossrefPubMedGoogle Scholar
  • 6 Thastrup O, Dawson AP, Scharff O, Foder B, Cullen PJ, Drøbak BK, Bjerrum PJ, Christensen SB, Hanely MR. Thapsigargin, a novel molecular probe for studying intracellular calcium release and storage. Agents Actions 1989; 27: 17-23
    CrossrefPubMedGoogle Scholar
  • 7 Papp B, Enyedi A, Kovács T, Sarkadi B, Wuytack F, Thastrup O, Gárdos G, Bredoux R, Levy-Tolenado S, Enouf J. Demonstration of two forms of calcium pumps by thapsigargin inhibition and radioimmunoblotting in platelet membrane vesicles. J Biol Chem 1991; 266: 14593-14596
    PubMedGoogle Scholar
  • 8 Brüne B, Ullrich V. Different calcium pools in human platelets and their role in thromboxane A2formation. J Biol Chem 1991; 266: 19232-19237
    PubMedGoogle Scholar
  • 9 Sargeant P, Clarkson WD, Sage SO, Heemskerk JWM. Calcium influx evoked by Ca2+store depletion in human platelets is more susceptible to cytochrome P-450 inhibitors than receptor-mediated calcium entry. Cell Calcium 1992; 13: 553-564
    CrossrefPubMedGoogle Scholar
  • 10 Heemskerk JWM, Vis PM, Feijge MAH, Hoyland J, Mason WT, Sage SO. Roles of phospholipase C and Ca2+-ATPase in calcium responses of single fibrinogen-bound platelets. J Biol Chem 1993; 268: 356-363
    PubMedGoogle Scholar
  • 11 Comfurius P, Senden JMG, Tilly RHJ, Schroit AJ, Bevers EM, Zwaal RFA. Loss of membrane phospholipid asymmetry in platelets and red cells may be associated with calcium-induced shedding of plasma membrane and inhibition of aminophospholipid translocase. Biochim Biophys Acta 1990; 1026: 153-160
    CrossrefPubMedGoogle Scholar
  • 12 Sims PJ, Faioni EM, Wiedmer T, Shattil SJ. Complement proteins C5b-9 cause release of membrane vesicles from the platelet surface that are enriched in the membrane receptor for coagulation Factor Va and express prothrombinase activity. J Biol Chem 1988; 263: 18205-18212
    PubMedGoogle Scholar
  • 13 Conner J, Pak CH, Zwaal RFA, Schroit AJ. Bidirectional transbilayer movement of phospholipid analogs in human red blood cells. J Biol Chem 1992; 267: 19412-19417
    PubMedGoogle Scholar
  • 14 Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+indicators with greatly improved fluorescent properties. J Biol Chem 1985; 260: 3440-3450
    PubMedGoogle Scholar
  • 15 Brüne B, Ullrich V. Calcium mobilization in human platelets by receptor agonists and calcium-ATPase inhibitors. FEBS Lett 1991; 284: 1-4
    CrossrefPubMedGoogle Scholar
  • 16 Schroit AJ, Zwaal RFA. Transbilayer movement of phospholipids in red cell and platelet membranes. Biochim Biophys Acta 1991; 1071: 313-329
    CrossrefPubMedGoogle Scholar
  • 17 Tilly RHJ, Senden JMG, Comfurius P, Bevers EM, zwaal RFA. Increased aminophospholipid translocase activity in human platelets during secretion. Biochim Biophys Acta 1990; 1029: 188-190
    CrossrefPubMedGoogle Scholar
  • 18 Malcolm KC, Fitzpatrick FA. Epoxyeicosatrienoic acids inhibit Ca2+entry into platelets stimulated by thapsigargin and thrombin. J Biol Chem 1992; 267: 19854-19858
    PubMedGoogle Scholar
  • 19 Rink TJ, Smith SW, Tsien RY. Cytoplasmic-free Ca2+in human platelets: Ca2+thresholds and Ca-independent activation for shape-change and secretion. FEBS Lett 1982; 148: 21-26
    CrossrefPubMedGoogle Scholar
  • 20 Chap HJ, Zwaal RFA, Van DeenenLLM. Action of highly purified phospholipases on blood platelets. Evidence for an asymmetric distribution of phospholipids in the surface membrane. Biochem Biophys Acta 1977; 467: 146-164
    CrossrefPubMedGoogle Scholar
  • 21 Schick PK, Kurica KB, Chacko GK. Location of phosphatidylethanolamine and phosphatidylserine in the human platelet plasma membrane. J Clin Invest 1976; 57: 1221-1226
    CrossrefPubMedGoogle Scholar
  • 22 Fox JEB, Austin CD, Boyles JK, Steffen PK. Role of the membrane skeleton in preventing the shedding of procoagulant-rich microvesicles from the platelet plasma membrane. J Cell Biol 1990; 111: 483-493
    CrossrefPubMedGoogle Scholar
  • 23 Sims PJ, Wiedmer T, Esmon CT, Weiss HJ, Shattil S. Assembly of the platelet prothrombinase complex is linked to vesiculation of the platelet plasma membrane. J Biol Chem 1989; 26: 17049-17057
    PubMedGoogle Scholar
  • 24 Bevers EM, Comfurius P, zwaal RFA. Platelet procoagulant activity: Physiological significance and mechanisms of exposure. Blood Rev 1991; 5: 146-154
    CrossrefPubMedGoogle Scholar
  • 25 Heemskerk JWM, Feijge MAH, Andree HAM, Sage SO. Function of intracellular [Ca2+]iin exocytosis and transbilayer movement in human platelets surface labeled with the fluorescent probe l-[4-trimethylammonio]phenyl)-6-phenyl-l,3,5-hexatriene. Biochim Biophys Acta 1993; 1147: 194-204
    CrossrefPubMedGoogle Scholar
  • 26 Bevers EM, Tilly RHJ, Senden JMG, Comfurius P, zwaal RFA. Exposure of endogenous phosphatidylserine at the outer surface of stimulated platelets is reversed by restoration of aminophopholipid translocase activity. Biochemistry 1989; 28: 2382-2387
    CrossrefPubMedGoogle Scholar
  • 27 Del PrincipeD, Menichelli A, De MatteisW, Di GiulioS, Giordani M, Savini I, Finazzi AgroA. Hydrogen peroxide is an intermediate in the platelet activation cascade triggered by collagen, but not by thrombin. Thromb Res 1991; 62: 365-375
    CrossrefPubMedGoogle Scholar