Platelets in patients with acute ischemic stroke are exhausted and refractory to thrombin, due to cleavage of the seven-transmembrane thrombin receptor (PAR-1)

 

 Buy Article Permissions and Reprints

Summary

Platelet activation is involved in the pathogenesis of cerebrovascular ischemia, but the major agonist involved has yet to be identified. To investigate the role of thrombin in platelet activation in patients with acute ischemic stroke, and while thrombin is the most likely candidate for activation of the thrombin receptor PAR-1 in vivo, we assessed its cleavage and internalization using the antibodies SPAN12, binding to uncleaved PAR-1, and WEDE15, recognizing cleaved and uncleaved, but not internalized PAR-1. In contrast to healthy age-matched controls, platelets from stroke patients exhibited significant cleavage and internalization of PAR-1 (P<0.001) and failed to respond to thrombin in vitro. Enhanced surface expression of CD62P, CD63, TSP-1 and less mepacrine uptake showed platelet degranulation during stroke. Platelets from patients with acute cerebral ischemia are exhausted and desensitized to thrombin through cleavage of PAR-1, indicating that high concentrations of thrombin occur with acute cerebrovascular ischemic events in vivo.

• References

• 1 Genton E, Barnett HJ, Fields WS. et al. Cerebral ischemia: the role of thrombosis and of antithrombotic therapy Study Group on Antithrombotic Therapy. Stroke 1977; 08: 150-75.
  CrossrefPubMedGoogle Scholar
• 2 Uchiyama S, Yamazaki M, Hara Y. et al. Alterations of platelet, coagulation, and fibrinolysis markers in patients with acute ischemic stroke. Semin Thromb Hemost 1997; 23: 535-41.
  Article in Thieme ConnectPubMedGoogle Scholar
• 3 Shah AB, Beamer N, Coull BM. Enhanced in vivo platelet activation in subtypes of ischemic stroke. Stroke 1985; 16: 643-7.
  CrossrefPubMedGoogle Scholar
• 4 Zeller JA, Tschoepe D, Kessler C. Circulating platelets show increased activation in patients with acute cerebral ischemia. Thromb Haemost 1999; 81: 373-7.
  Article in Thieme ConnectPubMedGoogle Scholar
• 5 Harker LA, Hanson SR. Platelet factors predisposing to arterial thrombosis. Baillière’s Clin Haematol 1994; 07: 499-522.
  PubMedGoogle Scholar
• 6 Sandercock P, Willems H. Medical treatment of acute ischaemic stroke. Lancet 1992; 339: 537-9.
  CrossrefPubMedGoogle Scholar
• 7 Berman CL, Yeo EL, Wencel-Drake JD. et al. Platelet alpha granule membrane protein that is associated with the plasma membrane after activation: characterization and subcellular localization of platelet activation-dependent granule external membrane protein. J Clin Invest 1986; 78: 130-7.
  CrossrefPubMedGoogle Scholar
• 8 Wolff R, Plow EF, Ginsberg MH. Interaction of thrombospondin with resting and stimulated human platelets. J Biol Chem 1986; 261: 6840-6.
  PubMedGoogle Scholar
• 9 Davey MG, Luscher EF. Actions of thrombin and other coagulant and proteolytic enzymes on blood platelets. Nature 1967; 216: 857-8.
  CrossrefPubMedGoogle Scholar
• 10 Kahn ML, Nakanishi-Matsui M, Shapiro MJ. et al. Protease-activated receptors 1 and 4 mediate activation of human platelets by thrombin. J Cin Invest 1999; 103: 879-87.
  CrossrefPubMedGoogle Scholar
• 11 Doermann D, Clemetson KJ, Kehrel BE. The GPIb thrombin-binding site is essential for thrombin-induced platelet procoagulant activity. Blood 2000; 96: 2469-78.
  PubMedGoogle Scholar
• 12 Brass LF, Vassallo Jr RR, Belmonte E. et al. Structure and function of the human platelet thrombin receptor: studies using monoclonal antibodies directed against a defined domain within the receptor N terminus. J Biol Chem 1992; 267: 13795-8.
  PubMedGoogle Scholar
• 13 Molino M, Bainton DF, Hoxie JA. et al. Thrombin receptors on human platelets: initial localization and subsequent redistribution during platelet activation. J Biol Chem 1997; 272: 6011-7.
  CrossrefPubMedGoogle Scholar
• 14 Kehrel BE, Wierwille S, Clemetson KJ. et al. Glycoprotein VI is a major collagen receptor for platelet activation: it recognizes the platelet-activating quaternary structure of collagen, whereas CD36, glycoprotein IIb/IIIa, and von Willebrand factor do not. Blood 1998; 91: 491-9.
  PubMedGoogle Scholar
• 15 Xia Z, Wong T, Liu Q. et al. Optimally functional fluorescein isothiocyanate-labelled fibrinogen for quantitative studies of binding to activated platelets and platelet aggregation. Br. J. Haematol 1996; 93: 204-14.
  CrossrefPubMedGoogle Scholar
• 16 Kehrel B, Kronenberg A, Schwippert B. et al. Thrombospondin binds normally to glycoprotein IIIb deficient platelets. Biochem Biophys Res Commun 1991; 179: 985-91.
  CrossrefPubMedGoogle Scholar
• 17 Michelson AD, Barnard MR, Hechtman HB. et al. In vivo tracking of platelets: circulating degranulated platelets rapidly lose surface Pselectin but continue to circulate and function. Proc Natl Acad Sci U S A 1996; 93: 11877-82.
  CrossrefPubMedGoogle Scholar
• 18 Wall JE, Buijs-Wilts M, Arnold JT. et al. A flow cytometric assay using mepacrine for study of uptake and release of platelet dense granule contents. Br J Haematol 1995; 89: 380-5.
  CrossrefPubMedGoogle Scholar
• 19 Iwamoto T, Kubo H, Takasaki M. Platelet activation in the cerebral circulation in different subtypes of ischemic stroke and Binswanger’s disease. Stroke 1995; 26: 52-6.
  CrossrefPubMedGoogle Scholar
• 20 McConnell JP, Cheryk LA, Durocher A. et al. Uriniary 11-dehydro-thromboxane B(2) and coagulation activation markers measured within 24 h of human acute ischemic stroke. Neurosci Lett 2001; 313: 88-92.
  CrossrefPubMedGoogle Scholar
• 21 Levine PH, Fisher M, Fullerton AL. et al. Human platelet factor 4: preparation from outdated platelet concentrates and application in cerebral vascular disease. Am J Hematol 1981; 10: 375-85.
  CrossrefPubMedGoogle Scholar
• 22 Grau AJ, Ruf A, Vogt A. et al. Increased fraction of circulating activated platelets in acute and previous cerebrovascular ischemia. Thromb Haemost 1998; 80: 298-301.
  Article in Thieme ConnectPubMedGoogle Scholar
• 23 Marquardt L, Ruf A, Mansmann U. et al. Course of platelet activation markers after ischemic stroke. Stroke 2002; 33: 2570-4.
  CrossrefPubMedGoogle Scholar
• 24 Meiklejohn DJ, Vickers MA, Morrison ER. et al. In vivo platelet activation in atherothrombotic stroke is not determined by polymorphisms of human platelet glycoprotein IIIa or Ib. Br J Haematol 2001; 112: 621-31.
  CrossrefPubMedGoogle Scholar
• 25 Furman MI, Benoit SE, Barnard MR. et al. Increased platelet reactivity and circulating monocyte-platelet aggregates in patients with stable coronary artery disease. J Am Coll Cardiol 1998; 31: 352-8.
  PubMedGoogle Scholar
• 26 Frijns CJM, Kappelle LJ, van Gijn J. et al. Souble adhesion molecules reflect endothelial cell activation in ischemic stroke and in carotid atherosclerosis. Stroke 1997; 28: 2214-8.
  CrossrefPubMedGoogle Scholar
• 27 Nieuwenhuis MJ, van Oosterhout JJG, Rozemuller E. et al. Studies with a monoclonal antibody against activated platelets: Evidence that a secreted 53,000-molecular weight lysosome-like granule protein is exposed on the surface on activated platelets in the circulation. Blood 1991; 78: 280-4.
  PubMedGoogle Scholar
• 28 Nishibori M, Cham B, McNicol A. et al. The protein CD63 is in platelet dense granules, is deficient in a patient with Hermansky-Pudlak syndrome, and appears identical to granulophysin. J Clin Invest 1993; 91: 1775-82.
  CrossrefPubMedGoogle Scholar
• 29 Woolkalis MJ, DeMelfi Jr TM, Blanchard N. et al. Regulation of thrombin receptors on human umbilical vein endothelial cells. J Biol Chem 1995; 270: 9868-75.
  CrossrefPubMedGoogle Scholar
• 30 Hammes SR, Coughlin SR. Protease-activated receptor-1 can mediate responses to SFLLRN in thrombin-desensitized cells: Evidence for a novel mechanism for preventing or terminating signaling by PAR1’s tethered ligand. Biochemistry 1999; 38: 2486-93.
  CrossrefPubMedGoogle Scholar
• 31 Kinlough-Rathbone RL, Packham MA, Guccione MA. et al. Characteristics of thrombin-degranulated human platelets: development of a method that does not use proteolytic enzmyes for deaggregation. Thromb Haemost 1991; 65: 403-10.
  Article in Thieme ConnectPubMedGoogle Scholar
• 32 Pulcinelli FM, Daniel JM, Riondino S. et al. Fibrinogen binding is independent of an increase in intracellular calcium concentration in thrombin degranulated platelets. Thromb Haemost 1995; 73: 304-308.
  Article in Thieme ConnectPubMedGoogle Scholar
• 33 De Candia E, De Cristofaro R, Landolfi R. Thrombin-induced platelet activation is inhibited by high and low-molecular-weight heparin. Circulation 1999; 99: 3308-14.
  CrossrefPubMedGoogle Scholar
• 34 Cunningham MA, Rondeau E, Chen X. et al. Protease-activated receptor 1 mediates thrombin-dependent, cell-mediated renal inflammation in crescentic glomerulonephritis. J Exp Med 2000; 191: 455-61.
  CrossrefPubMedGoogle Scholar
• 35 Parry MA, Myles T, Tschopp J. et al. Cleavage of the thrombin receptor: identification of potential activators and inactivators. Biochem J 1996; 320: 335-41.
  CrossrefPubMedGoogle Scholar
• 36 Pidard D, Renesto P, Berndt MC. et al. Neutrophil proteinase cathepsin G is proteolytically active on the human glycoprotein Ib- IX receptor: characterization of the cleavage sites within the glycoprotein Ib alpha subunit. Biochem J 1994; 303: 489-98.
  CrossrefPubMedGoogle Scholar
• 37 Pidard D, Renesto P, Rabhi S. et al. Regulation of the structure and activity of platelet adhesion receptors by leukocyte proteinases. Nouv Rev Fr Hématol 1994; 36 (Suppl. 01) S99-101.
  PubMedGoogle Scholar